CN101523156A - Colorimetric bio-barcode amplification assay for analyte detection - Google Patents
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Abstract
Description
Translated fromChinese相关申请的交叉参考Cross References to Related Applications
本申请要求2005年9月16日申请的U.S.临时专利申请No.60/717,851的优先权权益，在此以其整体通过引用并入本文，用于所有的目的。This application claims the benefit of priority to U.S. Provisional Patent Application No. 60/717,851, filed September 16, 2005, which is hereby incorporated by reference in its entirety for all purposes.
政府支持声明Statement of Government Support
本发明在依据合同No.DE-AC02-05CH11231在Lawrence Berkeley国家实验室得到U.S.能源部支持的工作进行过程中完成。政府在本发明中享有一定的权利。This invention was made in the course of work at Lawrence Berkeley National Laboratory with support from the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.
对序列表的参考References to Sequence Listings
本申请将所附的纸件和电子形式的序列表通过引用并入本文。The accompanying paper and electronic Sequence Listings are hereby incorporated by reference into this application.
发明背景Background of the invention
发明领域field of invention
本发明涉及一种灵敏的筛选方法，用于检测样品中一种或多种目标分析物的存在或不存在。特别地，本发明涉及利用报告寡核苷酸作为生化条形码用于检测溶液中的一种或多种分析物的方法。The present invention relates to a sensitive screening method for detecting the presence or absence of one or more target analytes in a sample. In particular, the present invention relates to methods for detecting one or more analytes in solution using reporter oligonucleotides as biochemical barcodes.
相关技术related technology
已经研发了多种高灵敏度的生物分子检测方法，但是很少实现了聚合酶链式反应(PCR)的灵敏度。生物条形码扩增测定是对于蛋白质和核酸目标物具有PCR-样灵敏度而不需要酶扩增的唯一生物检测方法。然而，目前的生物条形码检测方案仍然需要玻璃芯片上基于微阵列的寡核苷酸固定，表面钝化化学来最小化非特异性结合，芯片上固定的金纳米颗粒的银加强，光散射测量和定量步骤。发明人之一和其他人在US专利申请No.10/877,750，公开为US20050037397；U.S.专利申请No.10/788,414，公开为US20050009206和U.S.专利申请No.10/108211，授权为U.S.专利No.6,974,666中已经描述了这样的筛选方法和检测方案，在此将所有通过引用并入本文，用于所有的目的。A variety of highly sensitive biomolecular detection methods have been developed, but polymerase chain reaction (PCR) sensitivity has rarely been achieved. The biological barcode amplification assay is the only biological detection method with PCR-like sensitivity for protein and nucleic acid targets without the need for enzymatic amplification. However, current protocols for biological barcoding detection still require microarray-based immobilization of oligonucleotides on glass chips, surface passivation chemistry to minimize non-specific binding, silver enhancement of on-chip immobilized gold nanoparticles, light scattering measurements and quantification step. One of the inventors and others in US Patent Application No. 10/877,750, published as US20050037397; U.S. Patent Application No. 10/788,414, published as US20050009206 and U.S. Patent Application No. 10/108211, issued as U.S. Patent No. 6,974,666 Such screening methods and detection protocols have been described in , all of which are hereby incorporated by reference for all purposes.
重要地，复杂精密的设备如微阵列和芯片-显像工具限制了轻便性，并且测定成本必定是昂贵的。如果可以避免或最小化上述需求而没有牺牲生物条形码测定的渺(10-18)摩尔灵敏度，将是有利的。Importantly, sophisticated equipment such as microarrays and chip-imaging tools limit portability, and assays are inherently expensive. It would be advantageous if the above requirements could be avoided or minimized without sacrificing atto (10−18 ) molar sensitivity of biological barcoding assays.
本领域中的其他人已经描述了使用加盖寡核苷酸的金纳米颗粒探针的比色测定，包括Robert Elghanian等，Selective ColorimetricDetection of Polynucleotides Based on the Distance-DependentOptical Properties of Gold Nanoparticles，Science 1997年8月22日；277：1078-1081(在报告中)；James J.Storhoff等，One-PotColorimetric Differentiation of Polynucleotides with SingleBase Imperfections Using Gold Nanoparticle Probes，J.Am.Chem.Soc.；(文章)1998；120(9)；1959-1964。然而，基于金纳米颗粒的比色检测方法的典型检测极限是～nM。Colorimetric assays using gold nanoparticle probes capped with oligonucleotides have been described by others in the art, including Robert Elghanian et al., Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles, Science 1997 Aug 22;277:1078-1081 (in report); James J. Storhoff et al., One-PotColorimetric Differentiation of Polynucleotides with SingleBase Imperfections Using Gold Nanoparticle Probes, J.Am.Chem.Soc.; (Article) 1998; 120(9); 1959-1964. However, typical detection limits of gold nanoparticle-based colorimetric detection methods are ~nM.
生物条形码扩增测定已经成为检测整个样品中数十个至数百个生物目标物如蛋白质和核酸的强有力工具。然而，目前的生物条形码检测方案仍然需要许多实验步骤，包括玻璃芯片上基于微阵列的寡核苷酸固定，芯片上固定的金纳米颗粒的银加强和光散射测量。因此，需要研发一种能够最小化上述需求而获得渺摩尔灵敏度的生物条形码测定。Biological barcode amplification assays have emerged as powerful tools for the detection of tens to hundreds of biological targets such as proteins and nucleic acids throughout a sample. However, current protocols for biological barcode detection still require many experimental steps, including microarray-based immobilization of oligonucleotides on glass chips, silver enhancement of gold nanoparticles immobilized on the chip, and light scattering measurements. Therefore, there is a need to develop a biological barcoding assay that can achieve attomolar sensitivity while minimizing the above requirements.
发明概述Summary of the invention
本发明提供了检测样品中分析物的方法。在一个实施方案中，该方法包括提供怀疑含有目标分析物的样品，将多孔颗粒探针和磁性探针颗粒与样品接触，并使多孔颗粒探针和磁性探针颗粒两者结合目标分析物。多孔微粒探针包括特异性结合目标分析物的第一配体和生物条形码寡核苷酸。磁性纳米颗粒探针包括也特异性结合目标分析物的第二配体。如果样品中存在目标分析物，在目标分析物、多孔微粒探针和磁性纳米颗粒探针之间形成复合物。从样品分离出复合物，从复合物释放并收集条形码寡核苷酸，并检测条形码寡核苷酸。The present invention provides methods for detecting an analyte in a sample. In one embodiment, the method includes providing a sample suspected of containing an analyte of interest, contacting the porous particle probe and the magnetic probe particle with the sample, and allowing both the porous particle probe and the magnetic probe particle to bind the target analyte. The porous microparticle probe includes a first ligand that specifically binds an analyte of interest and a biological barcode oligonucleotide. The magnetic nanoparticle probe includes a secondary ligand that also specifically binds the analyte of interest. If the analyte of interest is present in the sample, a complex is formed between the analyte of interest, the porous microparticle probe, and the magnetic nanoparticle probe. The complex is isolated from the sample, the barcoded oligonucleotide is released and collected from the complex, and the barcoded oligonucleotide is detected.
在一些实施方案中，目标分析物是核酸，蛋白质，肽，金属离子，半抗原，药物，代谢物，杀虫剂或污染物。In some embodiments, the target analyte is a nucleic acid, protein, peptide, metal ion, hapten, drug, metabolite, pesticide, or pollutant.
在一些实施方案中，目标分析物是细胞因子。In some embodiments, the analyte of interest is a cytokine.
在一些实施方案中，目标分析物是趋化因子。In some embodiments, the analyte of interest is a chemokine.
在一些实施方案中，多孔微粒探针由包括聚苯乙烯，纤维素，硅石，氧化铁，聚丙烯酰胺，或各种多糖，葡聚糖，琼脂糖，纤维素，及其衍生物和组合的材料组成。In some embodiments, porous microparticle probes are made of polystyrene, cellulose, silica, iron oxide, polyacrylamide, or various polysaccharides, dextran, agarose, cellulose, and derivatives and combinations thereof. Material composition.
在一些实施方案中，用胺修饰多孔微粒探针。In some embodiments, porous microparticle probes are modified with amines.
在一些实施方案中，微粒具有约0.1微米至约5000微米的大小，优选约0.5微米至约10微米的大小，和更优选约3微米至约5微米的大小。In some embodiments, the microparticles have a size from about 0.1 microns to about 5000 microns, preferably from about 0.5 microns to about 10 microns, and more preferably from about 3 microns to about 5 microns.
在一些实施方案中，多孔微粒探针具有约50埃至约150埃的孔径大小，更优选约90埃至约110埃。In some embodiments, the porous microparticle probe has a pore size of about 50 Angstroms to about 150 Angstroms, more preferably about 90 Angstroms to about 110 Angstroms.
在一些实施方案中，多孔微粒探针具有约300m2/g至约500m2/g的表面积，更优选约400m2/g至约450m2/g。In some embodiments, the porous microparticle probe has a surface area of from about 300 m2 /g to about 500 m2 /g, more preferably from about 400 m2 /g to about 450 m2 /g.
在一些实施方案中，条形码寡核苷酸是基因、病毒RNA或DNA、细菌DNA、真菌DNA、哺乳动物DNA、cDNA、mRNA、RNA或DNA片段、天然或合成核酸或适体。In some embodiments, barcode oligonucleotides are genes, viral RNA or DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA or DNA fragments, natural or synthetic nucleic acids or aptamers.
在一些实施方案中，用可检测标记修饰条形码寡核苷酸。可检测标记可以是生物素，放射性标记，荧光标记，发色团，氧化还原活性基团，具有电特征(signature)的基团，催化基团或拉曼标记。In some embodiments, barcode oligonucleotides are modified with a detectable label. The detectable label can be biotin, a radioactive label, a fluorescent label, a chromophore, a redox active group, a group with an electrical signature, a catalytic group or a Raman label.
在一些实施方案中，条形码寡核苷酸和微粒是通用探针的成员。In some embodiments, barcoded oligonucleotides and microparticles are members of universal probes.
在一些实施方案中，配体是单克隆或多克隆抗体。In some embodiments, the ligand is a monoclonal or polyclonal antibody.
在一些实施方案中，通过比色测定进行条形码寡核苷酸的检测。在一些实施方案中，比色测定包括检测条形码寡核苷酸，通过提供包括第一和第二颗粒探针的溶液，其中第一颗粒探针包括与条形码寡核苷酸的一个末端互补的捕获寡核苷酸，并且其中第二颗粒探针包括与条形码寡核苷酸的另一相对末端互补的捕获寡核苷酸；将条形码寡核苷酸接触溶液并使条形码寡核苷酸与第一和第二颗粒探针杂交，由此第一和第二颗粒探针聚集成聚集物，其中溶液中的颜色改变表示所述聚集物的形成；并且检测所述溶液中的颜色改变。In some embodiments, detection of barcoded oligonucleotides is performed by a colorimetric assay. In some embodiments, the colorimetric assay comprises detection of a barcode oligonucleotide by providing a solution comprising a first and a second particle probe, wherein the first particle probe comprises a capture cap complementary to one end of the barcode oligonucleotide. oligonucleotide, and wherein the second particle probe comprises a capture oligonucleotide complementary to the other opposite end of the barcode oligonucleotide; contacting the barcode oligonucleotide with the solution and bringing the barcode oligonucleotide into contact with the first hybridizing to the second particle probe whereby the first and second particle probes aggregate into aggregates, wherein a color change in solution indicates formation of the aggregate; and detecting the color change in solution.
附图简述Brief description of the drawings
图1.比色生物条形码测定。A.胺修饰的多孔硅石珠子的探针制备和电子显微镜照片(插图)。B.白细胞介素-2检测方案。Figure 1. Colorimetric biological barcoding assay. A. Probe preparation and electron micrograph (inset) of amine-modified porous silica beads. B. Interleukin-2 assay protocol.
图2.点样在TLC平板上的金纳米颗粒聚集物的定量方法。点强度值与条形码DNA的数量成比例(聚集的金纳米颗粒越多，显现的颜色越浅)，并且条形码DNA的数量与存在的目标蛋白质的量成比例。Figure 2. Quantification of gold nanoparticle aggregates spotted on TLC plates. The spot intensity value is proportional to the amount of barcoded DNA (the more aggregated gold nanoparticles, the lighter the color appears), and the amount of barcoded DNA is proportional to the amount of target protein present.
图3.基于金纳米颗粒的比色条形码DNAIL-2检测(上：定量数据；下：TLC平板上金纳米颗粒点)。A.在缓冲液中。B.在人血清样品中。Figure 3. AuNP-based colorimetric barcoded DNAIL-2 detection (upper: quantitative data; lower: AuNP spot on TLC plate). A. In buffer. B. In human serum samples.
图4.多路比色生物条形码测定。A.显示了测定步骤的方案。B.测定中可以使用的多种类型的纳米颗粒。Figure 4. Multiplex colorimetric biological barcoding assay. A. Scheme showing the assay steps. B. Various types of nanoparticles that can be used in the assay.
发明详述Detailed description of the invention
I.介绍I. Introduction
本发明提供了简单的，超灵敏的生物条形码方法，用于检测目标分析物。出于以下原因，该生物条形码分析物检测方法是重要的。首先，该新方法已经显示出通过调节条形码探针的表面和大小可以显著提高每个探针的条形码DNA数量。这使得产生了多个实施方案来检测条形码DNA。在一个实施方案中，如实施例中所示的，使用了比色测定。其次，对于该测定的检测极限的数量级优于其他常规免疫测定。第三，该生物-条形码方法不需要复杂的仪器装置或试验步骤。探针溶液的简单混合和分离将获得渺摩尔灵敏度而不需要使用微阵列、复杂的信号扩增步骤如酶扩增和银加强或精密复杂的信号测量工具。因为读出是基于颜色改变，只需要最小的专门技术来进行测定。第四，研发了使用图形软件的定量方法用于定量比色条形码DNA检测测定，这对于使用之前的基于金纳米颗粒的比色DNA检测方案是不可能的。最后，该测定应当适于护理点(point-of-care)应用，只需要探针溶液和TLC平板。The present invention provides a simple, ultrasensitive method for biological barcoding for the detection of target analytes. This biological barcoded analyte detection method is important for the following reasons. First, the new method has been shown to significantly increase the amount of barcoded DNA per probe by tuning the surface and size of the barcoded probes. This has led to multiple embodiments for detecting barcoded DNA. In one embodiment, a colorimetric assay is used as shown in the Examples. Second, the detection limit for this assay is orders of magnitude superior to other conventional immunoassays. Third, the bio-barcoding method does not require complex instrumentation or experimental procedures. Simple mixing and separation of probe solutions will achieve attomolar sensitivity without the use of microarrays, complex signal amplification steps such as enzyme amplification and silver enhancement, or sophisticated signal measurement tools. Because the readout is based on a color change, only minimal expertise is required to make the determination. Fourth, a quantitative method using graphical software was developed for quantitative colorimetric barcoded DNA detection assays, which was not possible using previous gold nanoparticle-based colorimetric DNA detection protocols. Finally, the assay should be suitable for point-of-care applications requiring only probe solutions and TLC plates.
II.定义II. Definition
如整个说明书中所用的“条形码”，“生化条形码”，“生物条形码”，“条形码寡核苷酸”，“条形码DNA”，“DNA条形码”，“报告条形码”，“报告条形码DNA”等全部可以相互交替使用并具有相同的含义。条形码DNA可以是核酸，如脱氧核糖核酸或核糖核酸。优选，DNA条形码是预定序列的寡核苷酸。如果需要，可以将DNA条形码标记，例如，使用生物素，放射性标记或荧光标记。As used throughout the specification, "barcode", "biochemical barcode", "biological barcode", "barcode oligonucleotide", "barcode DNA", "DNA barcode", "reporter barcode", "reporter barcode DNA" etc. all can be used interchangeably and have the same meaning. The barcoded DNA can be a nucleic acid, such as deoxyribonucleic acid or ribonucleic acid. Preferably, the DNA barcode is an oligonucleotide of predetermined sequence. DNA can be barcoded if desired, for example, with biotin, radioactive or fluorescent labels.
术语“颗粒”指的是可以优选由金属，硅石，氧化硅或聚苯乙烯组成的小块物质。“颗粒”可以是任何形状的，如球形或杆状。如在此所用的术语“颗粒”特意包括纳米颗粒和微粒。The term "particles" refers to small pieces of material which may preferably consist of metal, silica, silicon oxide or polystyrene. "Particles" can be of any shape, such as spherical or rod-shaped. The term "particle" as used herein specifically includes nanoparticles and microparticles.
术语“复合物”或“探针复合物”或“颗粒复合物探针”指的是由包括报告寡核苷酸和目标分析物特异性配体的多孔微粒缀合包括对相同目标分析物特异性的配体的磁性探针颗粒构成的缀合物，使目标分析物结合于其上的两个配体。The term "complex" or "probe complex" or "particle complex probe" refers to a probe composed of a reporter oligonucleotide and a target analyte-specific ligand conjugated to the same target analyte. A conjugate composed of a magnetic probe particle with a specific ligand, allowing the target analyte to bind to the two ligands.
术语“分析物”，“目标分析物”或“目的分析物”指的是待检测的化合物或组合物，包括药物，代谢物，杀虫剂，污染物等。分析物可以由特异性结合对(sbp)的成员组成，并可以是配体，其是单价的(单表位(monoepitopic))或多价的(多表位(polyepitopic))，优选抗原或半抗原性的，且是单个化合物或多个化合物，其共享至少一个共同的表位或决定簇位点。分析物可以是细胞或微生物的一部分，细胞如细菌或带有血型抗原如A，B，D等或HLA抗原的细胞，微生物如细菌，真菌，原生动物或病毒。如果分析物是单表位的，可以将分析物进一步修饰，例如，在化学上修饰，以提供一个或多个另外的结合位点。在本发明的实践中，分析物具有至少两个结合位点。The term "analyte", "analyte of interest" or "analyte of interest" refers to a compound or composition to be detected, including drugs, metabolites, pesticides, pollutants and the like. The analyte may consist of members of a specific binding pair (sbp), and may be a ligand, which is monovalent (monoepitopic) or multivalent (polyepitopic), preferably an antigen or a half Antigenic, and is a single compound or a plurality of compounds that share at least one common epitope or determinant site. The analyte may be a part of a cell or microorganism, such as a bacterium or a cell bearing blood group antigens such as A, B, D etc. or HLA antigens, a microorganism such as bacteria, fungi, protozoa or viruses. If the analyte is monoepitopic, the analyte can be further modified, eg, chemically, to provide one or more additional binding sites. In the practice of the invention, the analyte has at least two binding sites.
术语“配体”指的是其受体天然存在或可以制备的任何有机化合物。术语配体还包括配体类似物，这是修饰的配体，通常是有机基团或分析物类似物，通常分子量高于100，其可以与类似配体竞争受体，修饰提供了一种将配体类似物连接另一个分子的方式。配体类似物通常不同于配体，差异不仅仅是用键替代了氢，键将配体类似物连接中枢或标记；但是不需要。配体类似物可以以与配体相似的方式结合受体。类似物可以是，例如，针对配体抗体的独特型的抗体。The term "ligand" refers to any organic compound whose receptors occur naturally or can be prepared. The term ligand also includes ligand analogs, which are modified ligands, usually organic moieties or analyte analogs, usually of molecular weight above 100, which can compete with similar ligands for receptors, the modification providing a The way a ligand analog attaches to another molecule. Ligand analogs often differ from ligands by not merely substituting bonds for hydrogens that attach ligand analogs to hubs or tags; but need not. Ligand analogs can bind receptors in a similar manner to ligands. An analog can be, for example, an antibody directed against an idiotype of the ligand antibody.
术语“受体”或“反配体”指的是能够识别分子的特定空间和极性组构的任何化合物或组合物，例如，表位或决定簇位点。说明性的受体包括天然存在的受体，例如，甲状腺素结合球蛋白，抗体，酶，Fab片段，凝集素，核酸，核酸适体，抗生物素蛋白，蛋白A，barsar，补体成分Clq等。抗生物素蛋白旨在包括蛋清抗生物素蛋白和来自其他来源的生物素结合蛋白，如链霉抗生物素蛋白。The term "receptor" or "antiligand" refers to any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, eg, an epitope or determinant site. Illustrative receptors include naturally occurring receptors such as thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, aptamers, avidin, protein A, barsar, complement component Clq, etc. . Avidin is intended to include egg white avidin and biotin binding proteins from other sources such as streptavidin.
术语“特异性结合对(sbp)成员”指的是两个不同分子中的一个，其特异性地结合另一个分子并可以限定为与另一个分子的特定空间和/或极性组构互补。特异性结合对的成员可以称为配体和受体(反配体)。这些通常是免疫对如抗原-抗体的成员，尽管其他特异性结合对，如生物素-抗生物素蛋白，酶-底物，酶-拮抗剂，酶-激动剂，药物-目标分子，激素-激素受体，核酸双链体，IgG-蛋白A/蛋白G，多核苷酸对如DNA-DNA，DNA-RNA，蛋白-DNA，脂质-DNA，脂质-蛋白，多糖-脂质，蛋白-多糖，核酸适体和相关的目标配体(例如，小的有机化合物，核酸，蛋白质，肽，病毒，细胞等)等不是免疫对，但包括于本发明和sbp成员的定义中。特异性结合对的成员可以是完整的分子，或只是分子的一部分，只要该成员特异性地结合目标分析物上的结合位点以形成特异性结合对。The term "specific binding pair (sbp) member" refers to one of two distinct molecules that specifically binds another molecule and can be defined as complementary to a particular spatial and/or polar organization of the other molecule. The members of a specific binding pair can be referred to as ligand and receptor (antiligand). These are usually members of immune pairs such as antigen-antibody, although other specific binding pairs such as biotin-avidin, enzyme-substrate, enzyme-antagonist, enzyme-agonist, drug-target molecule, hormone- Hormone receptors, nucleic acid duplexes, IgG-protein A/protein G, polynucleotide pairs such as DNA-DNA, DNA-RNA, protein-DNA, lipid-DNA, lipid-protein, polysaccharide-lipid, protein - Polysaccharides, aptamers and related target ligands (eg, small organic compounds, nucleic acids, proteins, peptides, viruses, cells, etc.) etc. are not immune pairs but are included in the invention and the definition of sbp members. A member of a specific binding pair can be an entire molecule, or only a portion of a molecule, so long as the member specifically binds to a binding site on the analyte of interest to form a specific binding pair.
术语“特异性结合”指的是与其他分子显著较低的识别相比较，两个不同分子中的一个对于另一个的特异性识别。通常，分子在其表面上或腔中具有一些区域，在两个分子间产生特异性识别。特异性结合的实例是抗体-抗原相互作用，酶-底物相互作用，多核苷酸相互作用等。The term "specific binding" refers to the specific recognition of one of two different molecules for the other compared to the significantly lower recognition of the other molecule. Typically, molecules have regions on their surfaces or in cavities that create specific recognition between two molecules. Examples of specific binding are antibody-antigen interactions, enzyme-substrate interactions, polynucleotide interactions, and the like.
如在此所用的，与另一个多核苷酸(或其互补链)最佳比对(含合适的核苷酸插入或缺失)时，多核苷酸或其片段与另一个是“实质上同源”(“实质上相似”)，使用BLASTN(Altschul，S.F.Gish，W.，Miller，W.，Myers，E.W.& Lipman，D.J.(1990)“Basiclocalalignment search tool.”J.Mol.Biol.215：403-410)，在至少约80％，优选至少约90％，更优选至少约95-98％核苷酸碱基中存在核苷酸序列同一性。为了确定两个不同多核苷酸之间的同源性，使用比对程序如BLASTN程序“BLAST 2 sequences”测定百分比同源性。对于公众使用可以通过互联网从国家生物技术信息中心(NCBI)获得该程序(Tatiana A.Tatusova，Thomas L.Madden(1999)，“Blast2 sequences-a new tool for comparing protein and nucleotidesequences”，FEMS Microbiol Lett.174：247-250)。可以使用的参数是产生最高计算的百分比同源性(如以下所计算的)的以下的任意组合，括号中显示了缺省参数：As used herein, a polynucleotide or fragment thereof is "substantially homologous" to another polynucleotide (or its complementary strand) when optimally aligned (containing insertions or deletions of appropriate nucleotides) to another polynucleotide (or its complementary strand). " ("substantially similar"), using BLASTN (Altschul, S.F. Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403 -410), the nucleotide sequence identity exists in at least about 80%, preferably at least about 90%, more preferably at least about 95-98% of the nucleotide bases. To determine the homology between two different polynucleotides, the percent homology is determined using an alignment program such as the BLASTN program "
程序-blastnprogram-blastn
配对奖赏-0或1(1)Matching Bonus - 0 or 1 (1)
错配惩罚-0，-1，-2或-3(-2)Mismatch penalty -0, -1, -2 or -3(-2)
开放缺口惩罚-0，1，2，3，4或5(5)Open Gap Penalty - 0, 1, 2, 3, 4 or 5 (5)
延伸缺口惩罚-0或1(1)Extended Gap Penalty - 0 or 1 (1)
缺口x_dropoff(减少)-0或50(50)nick x_dropoff (decrease) - 0 or 50 (50)
预期-10expected -10
字长-11word length - 11
滤器-低复杂性Filter - Low Complexity
术语“抗体”指的是特异性结合另一个分子并因此限定为与另一个分子的特定空间和极性组构互补的免疫球蛋白。抗体可以是单克隆或多克隆的，并可以通过本领域公知的技术来制备，如对宿主的免疫和血清的收集(多克隆的)或通过制备连续杂交细胞系并收集分泌的蛋白质(单克隆的)，或通过克隆和表达编码至少天然抗体特异性结合需要的氨基酸序列的核苷酸序列或其诱变形式。抗体可以包括完整的免疫球蛋白或其片段，所述免疫球蛋白包括各种类别和同种型，如IgA，IgD，IgE，IgG1，IgG2a，IgG2b和IgG3，IgM等。其片段可以包括Fab，Fv和F(ab’)2，Fab’等。此外，在合适的情况中，可以使用免疫球蛋白或其片段的聚集物，聚合物和缀合物，只要保持对特定分子的结合亲和性。The term "antibody" refers to an immunoglobulin that specifically binds another molecule and is thus defined as being complementary to a particular spatial and polar organization of the other molecule. Antibodies can be monoclonal or polyclonal and can be prepared by techniques well known in the art, such as immunization of a host and collection of serum (polyclonal) or by preparation of serial hybrid cell lines and collection of secreted protein (monoclonal) ), or by cloning and expressing nucleotide sequences encoding at least the amino acid sequences required for specific binding of native antibodies, or mutagenized versions thereof. Antibodies may include intact immunoglobulins or fragments thereof, including various classes and isotypes such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, and the like. Its fragments may include Fab, Fv and F(ab')2, Fab', etc. Furthermore, where appropriate, aggregates, polymers and conjugates of immunoglobulins or fragments thereof may be used so long as the binding affinity for the particular molecule is maintained.
III.检测样品中分析物的方法III. METHODS FOR DETECTING ANALYTES IN SAMPLES
现在参照图1B，本发明的一个实施方案提供了检测来自样品的目标分析物的方法。该方法包括提供怀疑含有目标分析物的样品，将多孔颗粒探针和磁性探针颗粒接触样品，并使多孔颗粒探针和磁性探针颗粒两者结合目标分析物。多孔颗粒(即，微粒或纳米颗粒)探针包括特异性结合目标分析物的第一配体和条形码寡核苷酸。磁性探针颗粒(即，纳米颗粒)包括也特异性结合目标分析物的第二配体。如果样品中存在目标分析物，在目标分析物、多孔颗粒探针和磁性探针颗粒之间形成复合物。从样品中分离出复合物，从复合物释放并收集条形码寡核苷酸，并检测条形码寡核苷酸。Referring now to FIG. 1B, one embodiment of the present invention provides a method of detecting an analyte of interest from a sample. The method includes providing a sample suspected of containing an analyte of interest, contacting a porous particle probe and a magnetic probe particle with the sample, and allowing both the porous particle probe and the magnetic probe particle to bind the target analyte. Porous particle (ie, microparticle or nanoparticle) probes include a first ligand that specifically binds an analyte of interest and a barcoded oligonucleotide. Magnetic probe particles (ie, nanoparticles) include a secondary ligand that also specifically binds the target analyte. If the target analyte is present in the sample, a complex is formed between the target analyte, the porous particle probe, and the magnetic probe particle. The complex is isolated from the sample, the barcoded oligonucleotide is released and collected from the complex, and the barcoded oligonucleotide is detected.
如图1B所示，将用配体官能化以捕获目标分析物的多孔微粒探针和磁性探针颗粒两者与怀疑含有目标分析物的样品混合。经混合，如果存在，目标分析物结合磁性探针颗粒和多孔微粒探针两者上的配体，以形成探针复合物，该探针复合物包括通过结合目标分析物的配体连接在一起的磁性探针颗粒和多孔微粒探针。As shown in Figure IB, both porous microparticle probes and magnetic probe particles functionalized with ligands to capture target analytes are mixed with a sample suspected of containing the target analyte. Upon mixing, if present, the target analyte binds to the ligands on both the magnetic probe particle and the porous microparticle probe to form a probe complex comprising magnetic probe particles and porous microparticle probes.
在一个实施方案中，该方法利用了目标分析物与用寡核苷酸标记的颗粒的结合事件和对那些结合事件的后续检测。在此所述方法的最后一步依赖于普通DNA的表面化学。因此，可以引入现有技术中颗粒DNA检测方法的许多高灵敏度方面，但允许检测多种生物分子如蛋白质，而非DNA-在检测事件过程中不存在蛋白质。对于表面测定，蛋白质通常比短的寡核苷酸更难以操作，因为它们易于呈现出对固体支持物更高的非特异性结合，这通常导致更高的背景信号。最后，对于同质测定，与这些纳米颗粒结构相关的异乎寻常敏锐的熔解曲线将使得与使用呈现出正常和宽DNA熔解行为的探针可能的情况相比较，设计更多的生物条形码。In one embodiment, the method utilizes binding events of target analytes to particles labeled with oligonucleotides and subsequent detection of those binding events. The final step of the method described here relies on the surface chemistry of ordinary DNA. Thus, many of the high-sensitivity aspects of prior art particle DNA detection methods can be incorporated, but allow the detection of a variety of biomolecules such as proteins, rather than DNA - without the protein being present during the detection event. For surface assays, proteins are generally more difficult to manipulate than short oligonucleotides because they tend to exhibit higher nonspecific binding to solid supports, which often results in higher background signals. Finally, for homogeneous assays, the unusually sharp melting curves associated with these nanoparticle structures will allow the design of more biological barcodes than is possible with probes that exhibit normal and broad DNA melting behavior.
本发明考虑了使用任何合适的颗粒，其上附有适用于检测测定的寡核苷酸。如在此所述的，每个微粒，磁性探针颗粒和纳米颗粒将具有多个与其连接的寡核苷酸。因此，每个颗粒-寡核苷酸缀合物可以结合多个具有互补序列的寡核苷酸或核酸。The present invention contemplates the use of any suitable particle to which oligonucleotides suitable for detection assays are attached. As described herein, each microparticle, magnetic probe particle and nanoparticle will have multiple oligonucleotides attached thereto. Thus, each particle-oligonucleotide conjugate can bind multiple oligonucleotides or nucleic acids with complementary sequences.
将寡核苷酸接触水溶液中的颗粒，持续足以使至少一些寡核苷酸通过官能团结合纳米颗粒的时间。可以根据经验来确定这样的时间。例如，已经发现约12至24小时的时间获得良好的结果。在一些临床检测的实施方案中，用于杂交的优选时间可为10分钟至12小时。也可以根据经验来确定用于寡核苷酸结合的其他合适条件。例如，约10-20nM的纳米颗粒浓度和室温温育获得了良好的结果。The oligonucleotides are contacted with the particles in the aqueous solution for a time sufficient for at least some of the oligonucleotides to bind to the nanoparticles through the functional groups. Such times may be determined empirically. For example, a time of about 12 to 24 hours has been found to give good results. In some clinical assay embodiments, the preferred time for hybridization may be 10 minutes to 12 hours. Other suitable conditions for oligonucleotide binding can also be determined empirically. For example, a nanoparticle concentration of about 10-20 nM and incubation at room temperature gave good results.
探针复合物形成后，从样品中分离出探针复合物。在优选的实施方案中，这通过磁性分离来进行，通过将样品暴露于磁场(例如，通过磁性分离装置)来促进磁性分离，磁场吸引探针复合物中的磁性颗粒并允许将其从样品中分离出来。因此，在本发明的一个方面中，颗粒探针复合物包括具有条形码寡核苷酸和配体的微粒，其中配体结合特定的目标分析物，并且目标分析物还结合磁性探针颗粒上的另一个配体。After the probe complexes are formed, the probe complexes are isolated from the sample. In preferred embodiments, this is performed by magnetic separation, facilitated by exposing the sample to a magnetic field (e.g., via a magnetic separation device), which attracts the magnetic particles in the probe complex and allows them to be removed from the sample. separate from. Therefore, in one aspect of the invention, the particle-probe complex comprises microparticles with a barcoded oligonucleotide and a ligand, wherein the ligand binds to a specific target analyte, and the target analyte also binds to the magnetic probe particle. Another ligand.
从样品中分离出来后，释放并捕获探针复合物中多孔微粒上连接的条形码寡核苷酸，用于进一步的检测或分析。对于其所连接的颗粒，通过破坏条形码与颗粒表面结合的化学释放剂来释放条形码。这样的试剂包括，但不限于，通过硫醇连接优先结合颗粒的任何分子，如其他含硫醇或二硫化物的分子，二硫苏糖醇(DTT)，二硫赤藓糖醇(DTE)，巯基乙醇等，和裂解二硫键并因此将条形码从其所连接的颗粒中释放出来的还原剂，如硼氢化钠。还可以通过将条形码暴露于使条形码与条形码通过其来连接颗粒的寡核苷酸去杂交(dehybridize)的条件，将条形码从颗粒中释放出来。After separation from the sample, the barcoded oligonucleotides attached to the porous microparticles in the probe complex are released and captured for further detection or analysis. For the particle to which it is attached, the barcode is released by disrupting the chemical release agent that binds the barcode to the particle surface. Such reagents include, but are not limited to, any molecule that preferentially binds to the particle via a thiol linkage, such as other thiol or disulfide-containing molecules, dithiothreitol (DTT), dithioerythritol (DTE) , mercaptoethanol, etc., and reducing agents, such as sodium borohydride, that cleave the disulfide bond and thus release the barcode from the particle to which it is attached. The barcode can also be released from the particle by exposing the barcode to conditions that dehybridize the barcode with the oligonucleotide through which the barcode is attached to the particle.
然后可通过任何合适的方式来检测条形码或报告寡核苷酸。通常，在检测之前，条形码通过去杂交从复合物中释放出来。可以使用任何合适的将条形码从复合物中去杂交并释放出来的溶液或介质。代表性的介质是水。The barcode or reporter oligonucleotide can then be detected by any suitable means. Typically, barcodes are released from the complex by dehybridization prior to detection. Any suitable solution or medium for dehybridizing and releasing the barcode from the complex may be used. A representative medium is water.
a.目标分析物a. Target analytes
目标分析物可以是核酸分子，蛋白质，肽，半抗原，金属离子，药物，代谢物，杀虫剂或污染物。该方法可以用于检测诸如以下的分析物的存在：毒素，激素，酶，凝集素，蛋白质，信号分子，无机或有机分子，抗体，污染物，病毒，细菌，其他病原生物体，独特型或其他细胞表面标记。意图本发明可以用于检测怀疑含有目标分析物的样品中的目标分析物的存在或不存在。Target analytes can be nucleic acid molecules, proteins, peptides, haptens, metal ions, drugs, metabolites, pesticides or pollutants. The method can be used to detect the presence of analytes such as: toxins, hormones, enzymes, lectins, proteins, signaling molecules, inorganic or organic molecules, antibodies, pollutants, viruses, bacteria, other pathogenic organisms, idiotypes or Other cell surface markers. It is intended that the present invention may be used to detect the presence or absence of a target analyte in a sample suspected of containing the target analyte.
在一些实施方案中，目标分析物由核酸组成，并且特异性结合互补物是寡核苷酸。或者，目标分析物是蛋白质或半抗原，并且特异性结合互补物是包括单克隆或多克隆抗体的抗体。或者，目标分析物是来自基因组DNA样品的序列，并且特异性结合互补物是寡核苷酸，该寡核苷酸具有与基因组序列的至少一部分互补的序列。基因组DNA可以是真核生物，细菌，真菌或病毒DNA。In some embodiments, the target analyte consists of nucleic acid and the specific binding complement is an oligonucleotide. Alternatively, the analyte of interest is a protein or hapten and the specific binding complement is an antibody including monoclonal or polyclonal antibodies. Alternatively, the target analyte is a sequence from a genomic DNA sample and the specific binding complement is an oligonucleotide having a sequence complementary to at least a portion of the genomic sequence. Genomic DNA can be eukaryotic, bacterial, fungal or viral DNA.
在一个实施方案中，特定细胞因子的检测可以用于癌症的诊断。特定的目标分析物包括细胞因子，如IL-2，如实施例中所示的。细胞因子是重要的目标分析物，因为细胞因子在造血作用的调节中起着关键作用；介导表型上不同的细胞的分化，迁移，活化和增殖。提高的细胞因子检测极限将使得可以更早和更准确地诊断和治疗癌症和免疫缺陷相关的疾病，并导致对细胞因子相关疾病和生物学的增加理解，因为当人受到外来抗原感染时，细胞因子是特征生物标记。In one embodiment, the detection of specific cytokines can be used in the diagnosis of cancer. Particular analytes of interest include cytokines, such as IL-2, as shown in the Examples. Cytokines are important target analytes because cytokines play a key role in the regulation of hematopoiesis; mediating differentiation, migration, activation and proliferation of phenotypically distinct cells. Improved cytokine detection limits will allow earlier and more accurate diagnosis and treatment of cancer and immunodeficiency-related diseases, and lead to an increased understanding of cytokine-related diseases and biology, because when a person is infected with a foreign antigen, cells Factors are characteristic biomarkers.
趋化因子是另一类重要的目标分析物。应答细菌感染，病毒和引起身体损伤的试剂如硅石或尿酸盐晶体而从多种细胞中释放出趋化因子。它们主要作为白细胞的化学引诱物，募集单核细胞，嗜中性粒细胞和其他效应细胞从血液至感染或损伤部位。它们由许多不同细胞类型来释放并用来指引先天性免疫中涉及的细胞以及获得性免疫系统的淋巴细胞。因此，提高的趋化因子检测极限将允许更早和更准确的诊断和治疗，即，对于细菌感染和病毒感染。Chemokines are another important class of target analytes. Chemokines are released from a variety of cells in response to bacterial infection, viruses and agents that cause physical damage such as silica or urate crystals. They primarily serve as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to the site of infection or injury. They are released by many different cell types and serve to direct cells involved in the innate immunity as well as lymphocytes of the adaptive immune system. Therefore, an improved limit of detection of chemokines will allow earlier and more accurate diagnosis and treatment, ie, for bacterial and viral infections.
在一些实施方案中，目标分析物可以是各种致病生物体，包括但不限于，唾液酸用来检测HIV，衣原体属(Chlamydia)，脑膜炎奈瑟菌(Neisseria meningitidis)，猪链球菌(Streptococcus suis)，沙门氏菌属(Salmonella)，腮腺炎、新城和各种病毒，包括呼肠孤病毒、仙台病毒和粘病毒；和9-OAC唾液酸用来检测冠状病毒，脑脊髓炎病毒和轮状病毒；非-唾液酸糖蛋白用来检测巨细胞病毒和麻疹病毒；CD4，血管活性肠肽和肽T用来检测HIV；表皮生长因子用来检测痘苗病毒；乙酰胆碱受体用来检测狂犬病；Cd3补体受体用来检测EB病毒；β-肾上腺素能受体用来检测呼肠弧病毒；ICAM-1，N-CAM和髓磷脂-相关糖蛋白MAb用来检测鼻病毒；脊髓灰质炎病毒受体用来检测脊髓灰质炎病毒；成纤维细胞生长因子受体用来检测疱疹病毒；寡聚甘露糖用来检测大肠杆菌；神经节苷脂GM1用来检测脑膜炎奈瑟菌；和抗体用来检测多种病原体(例如，淋病奈瑟菌(Neisseriagonorrhoeae)，创伤弧菌(V.vulnificus)，副溶血弧菌(V.parahaemolyticus)，霍乱弧菌(V.cholerae)和溶藻弧菌(V.alginolyticus))。In some embodiments, target analytes can be various pathogenic organisms, including but not limited to, sialic acid for detection of HIV, Chlamydia (Chlamydia), Neisseria meningitidis (Neisseria meningitidis), Streptococcus suis ( Streptococcus suis), Salmonella, mumps, Newcastle, and various viruses including reovirus, Sendai virus, and myxovirus; and 9-OAC sialic acid for the detection of coronaviruses, encephalomyelitis virus, and rotavirus Viruses; non-sialoglycoprotein for cytomegalovirus and measles virus; CD4, vasoactive intestinal peptide and peptide T for HIV; epidermal growth factor for vaccinia virus; acetylcholine receptor for rabies; Cd3 Complement receptors are used to detect Epstein-Barr virus; β-adrenergic receptors are used to detect Reovirus; ICAM-1, N-CAM and myelin-associated glycoprotein MAb are used to detect rhinovirus; The antibody was used to detect poliovirus; the fibroblast growth factor receptor was used to detect herpes virus; the oligomannose was used to detect E. coli; the ganglioside GM1 was used to detect Neisseria meningitidis; Detection of multiple pathogens (e.g., Neisseria gonorrhoeae (Neisseria gonorrhoeae), Vibrio vulnificus (V.vulnificus), Vibrio parahaemolyticus (V.parahaemolyticus), Vibrio cholerae (V.cholerae) and Vibrio alginolyticus (V. alginolyticus)).
在一些实施方案中，通过利用对不同目标分析物特异性的多个配体和利用对应于每个目标分析物的不同条形码寡核苷酸可检测多个目标分析物。In some embodiments, multiple target analytes can be detected by using multiple ligands specific for different target analytes and using different barcoded oligonucleotides corresponding to each target analyte.
b.样品b.sample
可以在样品中直接找到目标分析物，样品如来自宿主的体液。宿主可以是哺乳动物，爬行动物，鸟，两栖动物，鱼或昆虫。在优选的实施方案中，宿主是人。体液可以是，例如，尿液，血液，血浆，血清，唾液，精液，粪便，痰，脑脊液，泪液，粘液，脓，粘痰等。可以将颗粒与活细胞或含有活细胞的样品混合。Target analytes can be found directly in samples, such as bodily fluids from the host. The host can be a mammal, reptile, bird, amphibian, fish or insect. In preferred embodiments, the host is human. Bodily fluids can be, for example, urine, blood, plasma, serum, saliva, semen, feces, phlegm, cerebrospinal fluid, tears, mucus, pus, mucus, and the like. The particles can be mixed with living cells or samples containing living cells.
在样品是活细胞或含有活细胞的样品的情况中，细胞表面蛋白或其他分子可以作为目标分析物。这使得可以检测细胞活化和增殖事件，细胞相互作用，倍增(multiplexing)和其他生理相关事件。Where the sample is a living cell or a sample containing living cells, cell surface proteins or other molecules may serve as analytes of interest. This allows detection of cellular activation and proliferation events, cellular interactions, multiplexing and other physiologically relevant events.
c.多孔微粒探针c. Porous particle probe
在优选的实施方案中，本发明利用多孔微粒和基于金属纳米颗粒的比色DNA检测方案，用于直截了当的读出(图1)。在优选的实施方案中，多孔微粒探针应具有以下特征：捕获目标分析物的配体和条形码寡核苷酸，其是特异性条形码DNA序列。In a preferred embodiment, the present invention utilizes a porous microparticle and metal nanoparticle based colorimetric DNA detection scheme for straightforward readout (Figure 1). In a preferred embodiment, the porous microparticle probe should have the following features: a ligand to capture the analyte of interest and a barcode oligonucleotide, which is a specific barcode DNA sequence.
在一个实施方案中，微粒是具有限定孔隙度并由具有限定大小范围的小孔构成的多孔颗粒，其中将条形码寡核苷酸加于微粒的小孔内。使用多孔微粒可以使每个颗粒容纳数百万条形码DNA，因此使得可以使用具有渺摩尔灵敏度的比色条形码DNA检测方案。这是一个重要的进展，因为该方案具有生物条形码扩增方法的渺摩尔(10-18M)灵敏度以及基于金纳米颗粒的比色检测方法的简单，轻便和低成本。In one embodiment, the microparticles are porous particles of defined porosity and composed of pores of a defined size range, wherein barcoded oligonucleotides are incorporated within the pores of the microparticles. The use of porous microparticles allows the containment of millions of barcoded DNA per particle, thus enabling the use of colorimetric barcoded DNA detection protocols with attomolar sensitivity. This is an important advance because this protocol has the attomolar (10-18 M) sensitivity of biological barcode amplification methods and the simplicity, portability and low cost of gold nanoparticle-based colorimetric detection methods.
在一些实施方案中，多孔微粒探针可以由包括硅石和氧化硅的材料构成。如在此所用的术语“微粒”包括任何颗粒状珠子，球体，颗粒或载体，不管是生物可降解的或非生物可降解的，由天然存在的或合成的有机或无机多孔材料构成。特别地，微粒包括具有约0.1至约5000微米直径的任何颗粒状珠子，球体，颗粒或载体，更优选约1-5μm直径，更优选约3-4μm直径。如在此所用的术语“约”意思是包括多至所给范围±1单位。在另一个实施方案中，使用多孔硅石微粒(1.57×109ml-1直径：3.53±0.49μm)In some embodiments, porous particle probes may be constructed of materials including silica and silicon oxide. The term "microparticle" as used herein includes any granular bead, sphere, particle or carrier, whether biodegradable or non-biodegradable, composed of naturally occurring or synthetic organic or inorganic porous materials. In particular, microparticles include any particulate beads, spheres, particles or supports having a diameter of about 0.1 to about 5000 microns, more preferably about 1-5 microns in diameter, more preferably about 3-4 microns in diameter. As used herein, the term "about" is meant to include up to ± 1 unit of the given range. In another embodiment, porous silica microparticles (1.57×109 ml−1 diameter: 3.53±0.49 μm) are used
本发明的微粒由聚苯乙烯，硅石，氧化铁，聚丙烯酰胺和各种多糖包括葡聚糖，琼脂糖，纤维素，及其修饰的，交联的和衍生的实施方案构成。本发明微粒的特定实例包括聚苯乙烯，纤维素，与双丙烯酰胺交联的葡聚糖(Biogel.TM.，Bio-Rad，U.S.A.)，琼脂，玻璃珠子和乳胶珠子。衍生的微粒包括用羧基烷基如羧甲基，磷酰基和取代的磷酰基，硫酸盐，巯基和磺酰基，以及氨基和取代的氨基衍生的微粒。The microparticles of the present invention are constructed of polystyrene, silica, iron oxide, polyacrylamide, and various polysaccharides including dextran, agarose, cellulose, and modified, crosslinked, and derivatized embodiments thereof. Specific examples of microparticles of the present invention include polystyrene, cellulose, dextran cross-linked with bisacrylamide (Biogel.TM., Bio-Rad, U.S.A.), agar, glass beads and latex beads. Derivatized microparticles include those derivatized with carboxyalkyl groups such as carboxymethyl, phosphoryl and substituted phosphoryl, sulfate, mercapto and sulfonyl, and amino and substituted amino groups.
颗粒的大小，形状和化学组成将影响所得到的包括条形码DNA的探针的性质。这些性质包括光学性质，光电性质，电化学性质，电性质，在各种溶液中的稳定性，作为滤器时分离生物活性分子的能力等。考虑了使用具有不同大小，形状和/或化学组成的颗粒的混合物和使用具有均一大小，形状和化学组成的颗粒。The size, shape and chemical composition of the particles will affect the properties of the resulting probes comprising barcoded DNA. These properties include optical properties, optoelectronic properties, electrochemical properties, electrical properties, stability in various solutions, ability to separate biologically active molecules when used as a filter, and the like. The use of mixtures of particles of different size, shape and/or chemical composition and the use of particles of uniform size, shape and chemical composition are contemplated.
在一些实施方案中，微粒是氨基-官能化的，然后与配体和条形码寡核苷酸反应。在优选的实施方案中，多孔微粒探针由硅石和氧化铁构成并用胺基团官能化，用于用其他生物分子的进一步修饰。例如，可以从PHENOMENEX(Torrance，CA)获得这样的颗粒。其他人已经广泛使用了类似的戊二醛连接物化学来影响蛋白质与氨基官能化颗粒的连接。In some embodiments, microparticles are amino-functionalized and then reacted with ligands and barcode oligonucleotides. In a preferred embodiment, the porous microparticle probes are composed of silica and iron oxide and are functionalized with amine groups for further modification with other biomolecules. For example, such particles can be obtained from PHENOMENEX (Torrance, CA). Similar glutaraldehyde linker chemistry has been widely used by others to affect the attachment of proteins to amino-functionalized particles.
在另一个实施方案中，下文中所述的官能化纳米颗粒的方法可以用来官能化多孔微粒探针。在一些实施方案中，硅石覆盖的磁性颗粒是官能化的氨基-硅烷分子，以用胺来官能化硅石表面。In another embodiment, the methods described below for functionalizing nanoparticles can be used to functionalize porous microparticle probes. In some embodiments, the silica-covered magnetic particles are functionalized amino-silane molecules to functionalize the silica surface with amines.
多孔微粒影响可引入探针上的条形码寡核苷酸数量并因此影响灵敏度的其他性质包括：表面积，孔径大小，孔的互连性，亲水性和孔分布。Other properties of porous microparticles that affect the amount of barcoded oligonucleotides that can be introduced onto a probe and thus sensitivity include: surface area, pore size, interconnectivity of pores, hydrophilicity, and pore distribution.
i.表面积i. Surface area
通过调节条形码探针的表面和大小显著增加了每个探针的条形码寡核苷酸数量，这还形成了各种实施方案来检测多于一个条形码寡核苷酸。在生物条形码方法中，每个探针的条形码寡核苷酸数量是重要的，因为最终的检测信号与捕获的条形码DNA量成比例。The number of barcoded oligonucleotides per probe was significantly increased by adjusting the surface and size of the barcoded probes, which also resulted in various embodiments to detect more than one barcoded oligonucleotide. In biological barcoding methods, the number of barcoded oligonucleotides per probe is important because the final detection signal is proportional to the amount of barcoded DNA captured.
在一些实施方案中，多孔颗粒的表面积是约300m2/g至约500m2/g，更优选约400m2/g至约450m2/g。In some embodiments, the surface area of the porous particles is from about 300 m2 /g to about 500 m2 /g, more preferably from about 400 m2 /g to about 450 m2 /g.
在优选的实施方案中，大的探针大小(几微米)和孔隙率导致相对于过去的方法(没有孔的数十纳米的颗粒)显著提高的条形码寡核苷酸装载。使用UV-可见光谱学(对于单链DNA的UV吸收峰是在260nm)，测定出每～3.5微米珠子的平均条形码寡核苷酸总数为～3.6×106。与其他每个纳米颗粒探针只能载有数百个条形码DNA的基于纳米颗粒的条形码探针相比较，就每个条形码探针的条形码寡核苷酸数量而言，本发明的微粒导致扩增高出几个数量级。In a preferred embodiment, the large probe size (several microns) and porosity lead to significantly improved loading of barcoded oligonucleotides relative to past methods (particles of tens of nanometers without pores). Using UV-Vis spectroscopy (UV absorption peak at 260 nm for ssDNA), the average total number of barcoded oligonucleotides per ~3.5 micron bead was determined to be ~3.6 x106 . Compared to other nanoparticle-based barcode probes that can only carry hundreds of barcode DNA per nanoparticle probe, the microparticles of the present invention lead to amplification in terms of the number of barcode oligonucleotides per barcode probe. increased by several orders of magnitude.
ii.孔径大小ii. Aperture size
孔径大小也是多孔颗粒的一个重要方面。孔径大小必须足够大，使得条形码寡核苷酸在条形码与颗粒结合的过程中可以进入孔中，并在释放条形码寡核苷酸用于检测时离开孔。Pore size is also an important aspect of porous particles. The pore size must be large enough that the barcode oligonucleotide can enter the pore during the binding of the barcode to the particle and exit the pore when the barcode oligonucleotide is released for detection.
因此，在一些实施方案中，孔径大小是约50埃至约150埃，更优选约90埃至约110埃。Thus, in some embodiments, the pore size is from about 50 Angstroms to about 150 Angstroms, more preferably from about 90 Angstroms to about 110 Angstroms.
iii.互连性iii. Interconnectivity
多孔颗粒内的孔的互连性使得样品或流出物贯穿多孔颗粒流动。这些“通道”提供了制备并从孔内释放条形码DNA的方式。此外，通过具有通道，其防止了孔内的气穴形成，气穴可干扰条形码DNA进入和释放。The interconnectivity of the pores within the porous particle allows sample or effluent to flow through the porous particle. These "channels" provide the means by which the barcoded DNA is prepared and released from the wells. Furthermore, by having channels, it prevents the formation of air pockets within the pores, which can interfere with barcoded DNA entry and release.
因此，在优选的实施方案中，多孔颗粒具有通道来提供更大的条形码DNA容纳以及条形码DNA更好的结合和从颗粒释放。Thus, in preferred embodiments, the porous particles have channels to provide greater containment of barcoded DNA and better binding and release of barcoded DNA from the particle.
iv.亲水性iv. Hydrophilicity
在优选的实施方案中，多孔颗粒是亲水性的并极少至没有疏水性。亲水性多孔颗粒使得可以有效制备探针和有效释放条形码DNA用于检测。In preferred embodiments, the porous particles are hydrophilic and have little to no hydrophobicity. The hydrophilic porous particles allow efficient preparation of probes and efficient release of barcoded DNA for detection.
v.孔分布v. Pore distribution
在优选的实施方案中，多孔颗粒将具有可以引至颗粒上但不负面影响每个颗粒结构完整性的最大数量的孔。In preferred embodiments, porous particles will have the maximum number of pores that can be introduced onto the particle without adversely affecting the structural integrity of each particle.
每个颗粒的孔分布或孔数量还可以影响可容纳于颗粒上的条形码DNA数量。孔的数量对每个颗粒的表面积具有直接的影响。然而，颗粒可以具有的孔的数量存在限制。如果将太多的孔引至每个颗粒，可能损害颗粒的结构完整性。The pore distribution or number of pores per particle can also affect the amount of barcoded DNA that can be accommodated on the particle. The number of pores has a direct effect on the surface area per particle. However, there is a limit to the number of pores a particle can have. If too many pores are introduced into each particle, the structural integrity of the particle may be compromised.
d.配体d. Ligand
可以将连接用来捕获目标分析物的配体连接，可除去地连接，共价或非共价连接到多孔颗粒探针和磁性颗粒探针。Ligands attached to capture target analytes can be attached, removably attached, covalently or non-covalently attached to the porous particle probes and magnetic particle probes.
连接多孔颗粒探针的配体和连接磁性颗粒探针的配体都特异性地结合目标分析物。因此，在优选的实施方案中，目标分析物具有至少两个结合位点，使每个配体特异性地结合。Both the ligand attached to the porous particle probe and the ligand attached to the magnetic particle probe specifically bind the target analyte. Thus, in preferred embodiments, the analyte of interest has at least two binding sites to which each ligand specifically binds.
配体可以是将已知分析物作为特异性结合对成员的任何分子或物质。因此。特异性结合对的每个成员可以是核酸，寡核苷酸，肽核酸，多肽，抗原，碳水化合物，氨基酸，激素，类固醇，维生素，病毒，多糖，脂质，脂多糖，糖蛋白，脂蛋白，核蛋白，白蛋白，血红蛋白，凝血因子，肽激素，非肽激素，生物素，链霉抗生物素蛋白，细胞因子，趋化因子，包括肿瘤特异性表位的肽，细胞，细胞表面分子，微生物，小分子，酶，受体，通道，发色团，螯合化合物，磷酸反应基团，分子识别复合物，二硝基苯酚，电子供体或受体基团，疏水性化合物，亲水性化合物，有机分子和无机分子。A ligand can be any molecule or substance that has a known analyte as a member of a specific binding pair. therefore. Each member of a specific binding pair can be a nucleic acid, oligonucleotide, peptide nucleic acid, polypeptide, antigen, carbohydrate, amino acid, hormone, steroid, vitamin, virus, polysaccharide, lipid, lipopolysaccharide, glycoprotein, lipoprotein , nucleoproteins, albumin, hemoglobin, coagulation factors, peptide hormones, non-peptide hormones, biotin, streptavidin, cytokines, chemokines, peptides including tumor-specific epitopes, cells, cell surface molecules , microorganisms, small molecules, enzymes, acceptors, channels, chromophores, chelating compounds, phosphate reactive groups, molecular recognition complexes, dinitrophenols, electron donor or acceptor groups, hydrophobic compounds, hydrophilic Aqueous compounds, organic and inorganic molecules.
在一些实施方案中，配体是单克隆抗体或多克隆抗体，其中目标分析物是蛋白质，半抗原或肽。在抗体用作配体的情况中，通过使用不同的偶联化学，用来官能化磁性探针颗粒的抗体的表位不同于用来制备微粒探针的抗体的那些表位。因此，在优选的实施方案中，选择作为配体的抗体是具有两个不同表位的已经产生的抗体。对于重要的疾病标记，通过学术和商业方式可容易地获得许多具有不同表位的高质量抗体。而且，本领域公知可以由本领域技术人员产生针对配体的抗体。In some embodiments, the ligand is a monoclonal antibody or a polyclonal antibody, wherein the target analyte is a protein, hapten or peptide. In cases where antibodies are used as ligands, the epitopes of the antibodies used to functionalize the magnetic probe particles differ from those of the antibodies used to prepare the microparticle probes by using different coupling chemistries. Thus, in a preferred embodiment, the antibody selected as ligand is an antibody that has been raised with two different epitopes. For important disease markers, many high-quality antibodies with different epitopes are readily available through academic and commercial means. Furthermore, it is well known in the art that antibodies directed against the ligands can be generated by those skilled in the art.
在一些实施方案中，在目标分析物是核酸的情况中，配体是具有与该核酸的至少一部分序列互补的序列的寡核苷酸。In some embodiments, where the target analyte is a nucleic acid, the ligand is an oligonucleotide having a sequence complementary to at least a portion of the sequence of the nucleic acid.
在一些实施方案中，在目标分析物来自基因组DNA样品的情况中，配体是具有与基因组序列互补的序列的寡核苷酸。In some embodiments, where the target analyte is from a genomic DNA sample, the ligand is an oligonucleotide having a sequence complementary to the genomic sequence.
将氨基-官能化的磁性颗粒连接用于目标分析物的配体。在优选的实施方案中，在抗体用作配体的情况中，使用戊二醛-胺偶联化学，抗体的表位不同于用于制备条形码DNA的抗体的那些表位。Amino-functionalized magnetic particles are attached to ligands for target analytes. In a preferred embodiment, where antibodies are used as ligands, using glutaraldehyde-amine coupling chemistry, the epitopes of the antibodies are different from those of the antibodies used to prepare the barcoded DNA.
e.条形码寡核苷酸e. Barcoded oligonucleotides
在优选的实施方案中，可以连接，可除去地连接，共价或非共价连接条形码寡核苷酸，条形码寡核苷酸连接多孔微粒探针来捕获目标分析物。In preferred embodiments, barcode oligonucleotides can be attached, removably attached, covalently or non-covalently attached, barcode oligonucleotides attached to porous microparticle probes to capture target analytes.
可以使用将寡核苷酸连接至纳米球体表面上的任何合适方法。特别优选的将寡核苷酸连接至表面的方法是基于以下申请中所述的老化法：U.S.申请系列No.09/334,667，1999年6月25日申请；系列No.09/603,830，2000年6月26日申请；系列No.09/760,500，2001年1月12日申请；系列No.09/820,279，2001年3月28日申请；系列No.09/927,777，2001年8月10日申请；和国际申请no.PCT/US97/12783，1997年7月21日申请；PCT/US00/17507，2000年6月26日申请；PCT/US01/01190，2001年1月12日申请；PCT/US01/10071，2001年3月28日申请，在此将这些公开内容以其整体通过引用并入本文。老化法提供了具有出人意料的提高的稳定性和选择性的纳米颗粒-寡核苷酸缀合物。Any suitable method of attaching oligonucleotides to the surface of nanospheres can be used. A particularly preferred method of attaching oligonucleotides to surfaces is based on the aging method described in: U.S. Application Serial No. 09/334,667, filed June 25, 1999; Serial No. 09/603,830, issued in 2000 Filed June 26; Serial No. 09/760,500, filed January 12, 2001; Serial No. 09/820,279, filed March 28, 2001; Serial No. 09/927,777, filed August 10, 2001 and International Application nos. PCT/US97/12783, filed July 21, 1997; PCT/US00/17507, filed June 26, 2000; PCT/US01/01190, filed January 12, 2001; PCT/US00/17507, filed January 12, 2001; US01/10071, filed March 28, 2001, the disclosure of which is hereby incorporated by reference in its entirety. The aging method provides nanoparticle-oligonucleotide conjugates with unexpectedly improved stability and selectivity.
在一个实施方案中，该方法包括提供条形码寡核苷酸，该寡核苷酸优选具有与其共价结合的部分，该部分包括可以结合纳米颗粒的官能团。所述部分和官能团是允许寡核苷酸结合(即，通过化学吸附或共价结合)纳米颗粒的那些。例如，具有共价结合其5’或3’端的链烷硫醇，链烷二硫化物或环状二硫化物的寡核苷酸可以用来将寡核苷酸结合至多种纳米颗粒，包括金纳米颗粒。将寡核苷酸连接纳米颗粒的方法进一步描述于U.S.专利申请系列No.10/877,750中，以US20050037397公开，在此通过引用并入本文。In one embodiment, the method comprises providing a barcoded oligonucleotide, preferably having a moiety covalently bound thereto, the moiety comprising a functional group that can bind a nanoparticle. The moieties and functional groups are those that allow the oligonucleotide to bind (ie, by chemisorption or covalently bind) the nanoparticle. For example, oligonucleotides with alkanethiols, alkanedisulfides, or cyclic disulfides covalently bound to their 5' or 3' ends can be used to bind oligonucleotides to a variety of nanoparticles, including gold nanoparticles. Methods of linking oligonucleotides to nanoparticles are further described in U.S. Patent Application Serial No. 10/877,750, published as US20050037397, which is hereby incorporated by reference.
在一些实施方案中，条形码寡核苷酸通过连接物连接微粒。存在许多可购得的胺-反应性连接物(用于共价连接)。因此，考虑了通常用胺修饰微粒。优选，连接物进一步包括连接环状二硫化物的碳氢化合物部分。合适的碳氢化合物是可购得的，并将其连接环状二硫化物。优选，碳氢化合物部分是类固醇残基。已经出乎意料地发现与使用链烷硫醇或无环二硫化物作为连接物制备的缀合物相比较，使用包括连接环状二硫化物的类固醇残基的连接物制备的寡核苷酸颗粒缀合物对于硫醇(例如，聚合酶链式反应(PCR)溶液中所用的二硫苏糖醇)非常稳定。实际上，其他人已经发现本发明的寡核苷酸-颗粒缀合物稳定性高出300倍。参见U.S.专利申请系列No.10/877,750。该稳定性很可能是由于每个寡核苷酸通过两个硫原子而不是单个硫原子锚定到微粒这一事实。特别地，认为环状二硫化物的两个相邻硫原子具有螯合效应，这在稳定寡核苷酸-微粒缀合物中是有利的。连接物的大疏水性类固醇残基通过从水溶性分子接近纳米颗粒的表面筛选微粒也似乎有助于缀合物的稳定性。In some embodiments, barcoded oligonucleotides are attached to microparticles via linkers. There are many commercially available amine-reactive linkers (for covalent attachment). Therefore, modification of microparticles with amines is generally considered. Preferably, the linker further comprises a hydrocarbon moiety linking the cyclic disulfide. Suitable hydrocarbons are commercially available and linked to cyclic disulfides. Preferably, the hydrocarbon moiety is a steroid residue. It has been unexpectedly found that oligonucleotides prepared using linkers comprising steroid residues attached to cyclic disulfides compared to conjugates prepared using alkanethiols or acyclic disulfides as linkers The particle conjugates are very stable towards thiols such as dithiothreitol used in polymerase chain reaction (PCR) solutions. In fact, others have found that the oligonucleotide-particle conjugates of the present invention are 300-fold more stable. See U.S. Patent Application Serial No. 10/877,750. This stability is likely due to the fact that each oligonucleotide is anchored to the microparticle by two sulfur atoms rather than a single sulfur atom. In particular, two adjacent sulfur atoms of a cyclic disulfide are believed to have a chelating effect, which is advantageous in stabilizing oligonucleotide-microparticle conjugates. The large hydrophobic steroid residue of the linker also appears to contribute to the stability of the conjugate by screening microparticles from water-soluble molecules approaching the nanoparticle surface.
在另一个实施方案中，使用基于硫的官能团将条形码寡核苷酸结合到微粒。U.S.专利申请系列No.09/760,500和09/820,279和国际申请no.PCT/US01/01190和PCT/US01/10071描述了本发明实践中有用的用环状二硫化物官能化的寡核苷酸。环状二硫化物优选在其环中具有5或6个原子，包括两个硫原子。合适的环状二硫化物是可购得的，或可以通过已知程序合成。还可以使用环状二硫化物的还原形式。In another embodiment, sulfur-based functional groups are used to attach barcode oligonucleotides to microparticles. U.S. Patent Application Serial Nos. 09/760,500 and 09/820,279 and International Application nos. PCT/US01/01190 and PCT/US01/10071 describe cyclic disulfide-functionalized oligonucleotides useful in the practice of the invention . The cyclic disulfide preferably has 5 or 6 atoms in its ring, including two sulfur atoms. Suitable cyclic disulfides are commercially available or can be synthesized by known procedures. Reduced forms of cyclic disulfides can also be used.
在一个实施方案中，使用乙醇胺来钝化微粒上所有未反应的反应位点。还可以另外或替代地使用如牛血清白蛋白的蛋白质来进一步钝化微粒表面上没有活性的区域。In one embodiment, ethanolamine is used to deactivate all unreacted reactive sites on the microparticles. Proteins such as bovine serum albumin may additionally or alternatively be used to further passivate inactive areas on the surface of the microparticles.
如定义中所述的，DNA条形码可以是核酸，如脱氧核糖核酸或核糖核酸。优选，DNA条形码是预定序列的寡核苷酸。DNA条形码寡核苷酸可以包括基因；病毒RNA和DNA；细菌DNA；真菌DNA；哺乳动物DNA，cDNA，mRNA，RNA和DNA片段；寡核苷酸；合成的寡核苷酸；修饰的核苷酸；单链和双链核酸；天然和合成的核酸；和适体。As stated in the definitions, a DNA barcode may be a nucleic acid, such as deoxyribonucleic acid or ribonucleic acid. Preferably, the DNA barcode is an oligonucleotide of predetermined sequence. DNA barcoding oligonucleotides can include genes; viral RNA and DNA; bacterial DNA; fungal DNA; mammalian DNA, cDNA, mRNA, RNA and DNA fragments; oligonucleotides; synthetic oligonucleotides; modified nucleosides acids; single- and double-stranded nucleic acids; natural and synthetic nucleic acids; and aptamers.
制备预定序列寡核苷酸的方法是公知的。参见，例如，Sambrook等，Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)(第2版，1989)和F.Eckstein(编辑)Oligonucleotides andAnalogues(寡核苷酸和类似物)，第1版(Oxford University Press，New York，1991)。固相合成方法对于寡核糖核苷酸和寡脱氧核糖核苷酸都是优选的(合成DNA的公知方法也可用于合成RNA)。还可以通过酶制备寡核苷酸。对于具有其所结合的目标分析物的特异性结合互补物的寡核苷酸，可以使用将特异性结合互补物如蛋白质与寡核苷酸连接的任何合适方法。Methods for preparing oligonucleotides of predetermined sequence are well known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st ed. (Oxford University Press, New York, 1991). Solid phase synthesis methods are preferred for both oligoribonucleotides and oligodeoxyribonucleotides (well known methods for synthesizing DNA can also be used for synthesizing RNA). Oligonucleotides can also be prepared enzymatically. For oligonucleotides having a specific binding complement for an analyte of interest to which they bind, any suitable method of linking a specific binding complement, such as a protein, to the oligonucleotide may be used.
本发明考虑了使用通过本领域技术人员已知的技术设计的序列，包括，优化退火温度，序列对模板的特异性和序列的长度。可以使用引物预测软件如Oligo6(Molecular Biology Insights，Inc.，Cascade，CO)进行序列的设计。还可以使用用于引物设计的定制脚本和软件。The present invention contemplates the use of sequences designed by techniques known to those skilled in the art, including, optimization of annealing temperature, sequence-to-template specificity, and sequence length. The sequence can be designed using primer prediction software such as Oligo6 (Molecular Biology Insights, Inc., Cascade, CO). Custom scripts and software for primer design are also available.
任何独特的寡核苷酸序列及其互补序列可以用于条形码寡核苷酸。优选用作条形码寡核苷酸的寡核苷酸序列在严谨条件下与其互补序列杂交。如在此所用的术语“严谨条件”指的是序列将与其目标子序列或互补物杂交而不与其他序列杂交的条件。严谨条件是序列依赖性的且在不同情况下将不同。较长的序列在较高的温度特异性地杂交。通常，选择严谨条件为比在限定离子强度和pH下特定序列的热熔点(Tm)低约15℃。Tm是平衡时50％与目标序列互补的探针与目标序列杂交的温度(在限定的离子强度，pH和核酸浓度下)。(因为目标序列通常过量存在，因此在Tm，平衡时50％探针被占据)。Any unique oligonucleotide sequence and its complement can be used for barcoding oligonucleotides. Preferably, the oligonucleotide sequence used as a barcode oligonucleotide hybridizes to its complement under stringent conditions. The term "stringent conditions" as used herein refers to conditions under which a sequence will hybridize to its target subsequence or complement, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 15°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (Because the target sequence is usually present in excess, 50% of the probe is occupied at Tm, equilibrium).
在一些实施方案中，用可检测标记修饰条形码寡核苷酸。可检测标记的实例包括生物素，放射性标记，荧光标记，发色团，氧化还原活性基团，具有电特征的基团，催化基团和拉曼标记。In some embodiments, barcode oligonucleotides are modified with a detectable label. Examples of detectable labels include biotin, radioactive labels, fluorescent labels, chromophores, redox active groups, groups with electrical characteristics, catalytic groups and Raman labels.
可以在以下文献中找到这些特异性条形码DNA序列的实例，例如，Multiplexed Detection of Protein Cancer Markers withBiobarcoded Nanoparticle Probes，Stoeva等，128J.Am.Chem.Soc.8378-8379(2006)；Bio-Bar-Code-Based DNADetection with PCR-like Sensitivity，Nam等126J.Am.Chem.Soc.5932-5933(2004)；和Multiplexed DNA Detectionwith Biobarcoded Nanoparticle Probes，Soteva等，45 Angew.Chem.Int.Ed.，3303-3306(2006)，在此通过引用并入本文。Examples of these specific barcode DNA sequences can be found in, for example, Multiplexed Detection of Protein Cancer Markers with Biobarcoded Nanoparticle Probes, Stoeva et al., 128 J.Am.Chem.Soc.8378-8379 (2006); Bio-Bar-Code -Based DNA Detection with PCR-like Sensitivity, Nam et al. 126 J.Am.Chem.Soc.5932-5933 (2004); and Multiplexed DNA Detection with Biobarcoded Nanoparticle Probes, Soteva et al., 45 Angew.Chem.Int.Ed., 3303-3306 (2006), incorporated herein by reference.
在优选的实施方案中，条形码DNA是具有限定序列的3’氨基-官能化的条形码DNA互补物(例如，作为鉴定标签)，来鉴定微粒，用于检测特定目标分析物，因此可以检测样品中的多个目标分析物。In preferred embodiments, the barcode DNA is a 3' amino-functionalized barcode DNA complement with a defined sequence (e.g., as an identification tag) to identify the microparticle for the detection of a specific target analyte, thereby enabling the detection of multiple target analytes.
在一个实施方案中，该方法利用作为生化条形码的寡核苷酸，用于检测样品中的单个或多个分析物。该方法利用用纳米颗粒直接或间接官能化的识别元件(例如，蛋白质或核酸)和之前观察到导致金纳米颗粒聚集的杂交事件可以显著改变它们的物理特性(例如，光学，电学，机械)。总的构思是每个识别元件可以结合不同的寡核苷酸序列(即，DNA条形码)，这些序列具有离散的和可裁制的杂交和熔解特性以及与纳米颗粒相关的物理信号，所述纳米颗粒在熔解时改变以解码多分析物测定中的一系列分析物。因此，可以使用DNA相连聚集物的熔解温度以及与纳米颗粒相关的物理特性，所述纳米颗粒在熔解时改变以解码多分析物测定中的一系列分析物。在此的条形码不同于基于物理诊断标记物的那些，如纳米棒，荧光团标记的珠子和量子点，因为解码信息是存储于预先设计的寡核苷酸序列中的化学信息形式。In one embodiment, the method utilizes oligonucleotides as biochemical barcodes for the detection of single or multiple analytes in a sample. This approach exploits the direct or indirect functionalization of recognition elements (e.g., proteins or nucleic acids) with nanoparticles and the previous observation that hybridization events leading to aggregation of gold nanoparticles can significantly alter their physical properties (e.g., optical, electrical, mechanical). The general idea is that each recognition element can incorporate distinct oligonucleotide sequences (i.e., DNA barcodes) with discrete and tailorable hybridization and melting properties and physical signals associated with nanoparticles that The particles change upon melting to decode a range of analytes in a multi-analyte assay. Thus, the melting temperature of DNA-linked aggregates and the physical properties associated with nanoparticles that change upon melting can be used to decode a range of analytes in a multi-analyte assay. Barcodes here differ from those based on physical diagnostic markers, such as nanorods, fluorophore-labeled beads, and quantum dots, because the decoded information is in the form of chemical information stored in predesigned oligonucleotide sequences.
f.磁性探针颗粒f. Magnetic probe particles
磁性探针颗粒可以由包括氧化铁和其他铁磁材料的磁性材料构成。可以用硅石或聚合物如聚丙烯酰胺，聚苯乙烯等包被磁性探针颗粒，如对于多孔微粒所述的对其表面官能化。Magnetic probe particles may be composed of magnetic materials including iron oxide and other ferromagnetic materials. Magnetic probe particles can be coated with silica or polymers such as polyacrylamide, polystyrene, etc., and their surface functionalized as described for porous particles.
在优选的实施方案中，磁性探针颗粒可以是具有约0.1纳米至约5000微米直径的纳米颗粒或微粒。合适的磁性颗粒广泛用于本领域中，并可以从供应商如Dynal Biotech(新近被Invitrogen获得)获得。In preferred embodiments, the magnetic probe particles may be nanoparticles or microparticles having a diameter of about 0.1 nanometers to about 5000 micrometers. Suitable magnetic particles are widely used in the art and are available from suppliers such as Dynal Biotech (recently acquired by Invitrogen).
在一个实施方案中，按照实施例中所述的使用戊二醛-胺偶联化学来制备磁性颗粒。In one embodiment, magnetic particles were prepared using glutaraldehyde-amine coupling chemistry as described in the Examples.
本发明实践中有用的微粒和纳米颗粒包括金属(例如，金，银，铜和铂)，半导体(例如，CdSe，CdS以及用ZnS覆盖的CdS或CdSe)和磁性(例如，铁磁)胶体物质。本发明实践中有用的其他纳米颗粒包括ZnS，ZnO，TiO2，AgI，AgBr，HgI2，PbS，PbSe，ZnTe，CdTe，In2S3，In2Se3，Cd3P2，Cd3As2，InAs和GaAS。纳米颗粒的大小优选为约5nm至约150nm(平均直径)，更优选约5至约50nm，最优选约10至约30nm。纳米颗粒还可以是棒状，棱柱或四面体。Microparticles and nanoparticles useful in the practice of the present invention include metallic (e.g., gold, silver, copper, and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe capped with ZnS) and magnetic (e.g., ferromagnetic) colloidal species . Other nanoparticles useful in the practice of this invention include ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs and GaAS. The size of the nanoparticles is preferably from about 5 nm to about 150 nm (average diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. Nanoparticles can also be rod-shaped, prismatic or tetrahedral.
制备金属，半导体和磁性纳米颗粒的方法是本领域公知的。参见，例如，Schmid，G.(编辑)Cluster and Colloids(VCH，Weinheim，1994)；Hayat，M.A.(编辑)Colloidal Gold：Principles，Methods，and Applications(Academic Press，San Diego，1991)；Massart，R.，IEEE Taransactions On Magnetics，17，1247(1981)；Ahmadi，T.S.等，Science，272，1924(1996)；Henglein，A.等，J.Phys.Chem.，99，14129(1995)；Curtis，A.C.等，Angew.Chem.Int.Ed.Engl.，27，1530(1988)。Methods of preparing metallic, semiconducting and magnetic nanoparticles are well known in the art. See, eg, Schmid, G. (ed.) Cluster and Colloids (VCH, Weinheim, 1994); Hayat, M.A. (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R. ., IEEE Taransactions On Magnetics, 17, 1247(1981); Ahmadi, T.S. et al., Science, 272, 1924(1996); Henglein, A. et al., J.Phys.Chem., 99, 14129(1995); Curtis, A.C. et al., Angew. Chem. Int. Ed. Engl., 27, 1530 (1988).
制备ZnS，ZnO，TiO2，AgI，AgBr，HgI2，PbS，PbSe，ZnTe，CdTe，In2S3，In2Se3，Cd3P2，Cd3As2，InAs和GaAS纳米颗粒的方法也是本领域已知的。参见，例如，Weller，Angrew.Chem.Int.Ed.Engl.，32，41(1993)；Henglein，Top.Curr.Chem.，143，113(1988)；Henglein，Chem.Rev.，89，1861(1989)；Brus，Appl.Phys.A.，53，465(1991)；Bahncmann，Photochemical Conversion and Storageof Solar Energy(Pelizetti和Schiavello编辑，1991)，p251；Wang和Herron，J.Phys.Chem.，95，525(1991)；Olshavsky等，J.Am.Chem.Soc.，112，9438(1990)；Ushida等，J.Phys.Chem.，95，5382(1992)。Methods of preparing ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs and GaAS nanoparticles are also known in the art. See, e.g., Weller, Angrew. Chem. Int. Ed. Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); (1989); Brus, Appl. Phys. A., 53, 465 (1991); Bahncmann, Photochemical Conversion and Storage of Solar Energy (eds. Pelizetti and Schiavello, 1991), p251; Wang and Herron, J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).
也可以从例如Ted Pella，Inc.(金)，Amersham Corporation(金)和Nanoprobes，Inc.(金)购得合适的纳米颗粒。Suitable nanoparticles are also commercially available from, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
目前优选用于检测核酸的是金纳米颗粒。金胶体颗粒对于产生其漂亮颜色的条带具有高消光系数。这些强烈的颜色随着颗粒大小，浓度，颗粒间的距离以及聚集程度和聚集物形状(几何学)而改变，使这些材料对于比色测定特别有吸引力。例如，连接金纳米颗粒的寡核苷酸与寡核苷酸和核酸的杂交形成肉眼可见的立即颜色改变。Presently preferred for the detection of nucleic acids are gold nanoparticles. Gold colloidal particles have a high extinction coefficient for bands that produce their beautiful colors. These intense colors vary with particle size, concentration, distance between particles, and degree of aggregation and aggregate shape (geometry), making these materials particularly attractive for colorimetric assays. For example, hybridization of oligonucleotides attached to gold nanoparticles to oligonucleotides and nucleic acids results in an immediate color change visible to the naked eye.
将颗粒或寡核苷酸或两者官能化，以便将寡核苷酸连接到颗粒。这样的方法是本领域已知的。例如，用链烷硫醇在其3’-端或5’-端官能化的寡核苷酸容易地连接金纳米颗粒。参见Whitesides，Proceedings of the Robert A.Welch Foundation 39th Conferenceon Chemical Research Nanophase Chemistry，Houston，Tex.，p109-121(1995)。还可以参见，Mucic等，Chem.Commun.555-557(1996)(描述了将3’硫醇DNA连接到平的金表面的方法；该方法可以用于将寡核苷酸连接纳米颗粒)。链烷硫醇方法也可以用于将寡核苷酸连接其他金属，半导体和磁性胶体以及连接上述的其它纳米颗粒。用于将寡核苷酸连接到固体表面的其他官能团包括硫代磷酸基团(参见，例如，U.S.专利No.5,472,881，用于寡核苷酸-硫代磷酸酯与金表面的结合)，取代的烷基硅氧烷(参见，例如，Burwell，ChemicalTechnology，4，370-377(1974)以及Matteucci和Caruthers，J.Am.Chem.Soc.，103，3185-3191(1981)，关于寡核苷酸与硅石和玻璃表面的结合，和Grabar等，Anal.Chem.，67，735-743，关于氨基烷基硅氧烷的结合和巯基烷基硅氧烷的类似结合)。末端为5’硫代核苷或3’硫代核苷的寡核苷酸也可以用于连接寡核苷酸到固体表面。以下的参考文献描述了可以用来连接寡核苷酸到纳米颗粒的其他方法：Nuzzo等，J.Am.Chem.Soc.，109，2358(1987)(金上面的二硫化物)；Allara和Tompkins，Langmuir，1，45(1985)(铝上面的羧酸)；Allara和Tompkins，J.Colloid Interface Sci.，49，410-421(1974)(铜上面的羧酸)；Iler，The Chemistry Of Silica，第6章，(Wiley 1979)(硅石上的羧酸)；Timmons和Zisman，J.Phys.Chem.，69，984-990(1965)(铂上面的羧酸)；Soriaga和Hubbard，J.Am.Chem.Soc.，104，3937(1982)(铂上面的芳香环化合物)；Hubbard，Acc.Chem.Res.，13，177(1980)(铂上面的环丁砜，亚砜和其他官能化的溶剂)；Hickman等，J.Am.Chem.Soc.，111，7271(1989)(铂上面的异腈)；Maoz和Sagiv，Langmuir，3，1045(1987)(硅石上的硅烷)；Maoz和Sagiv，Langmuir，3，1034(1987)(硅石上的硅烷)；Wasserman等，Langmuir，5，1074(1989)(硅石上的硅烷)；Eltekova和Eltekov，Langmuir，3，951(1987)(二氧化钛和硅石上的芳香羧酸，醛，醇和甲氧基)；Lee等，J.Phys.Chem.，92，2597(1988)(金属上的刚性磷酸盐/酯)。Either the particle or the oligonucleotide or both are functionalized in order to attach the oligonucleotide to the particle. Such methods are known in the art. For example, gold nanoparticles are readily attached to oligonucleotides functionalized with alkanethiols at their 3'- or 5'-ends. See Whitesides, Proceedings of the Robert A. Welch Foundation 39th Conference on Chemical Research Nanophase Chemistry, Houston, Tex., pp 109-121 (1995). See also, Mucic et al., Chem. Commun. 555-557 (1996) (describing a method for attaching 3' thiol DNA to a flat gold surface; this method can be used to attach oligonucleotides to nanoparticles). The alkanethiol approach can also be used to attach oligonucleotides to other metals, semiconductors, and magnetic colloids as well as to other nanoparticles mentioned above. Other functional groups used to attach oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Patent No. 5,472,881 for oligonucleotide-phosphorothioate binding to gold surfaces), substituted (see, for example, Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J.Am.Chem.Soc., 103, 3185-3191 (1981), on oligonucleotides binding of acids to silica and glass surfaces, and Grabar et al., Anal. Chem., 67, 735-743, on the binding of aminoalkylsiloxanes and similar binding of mercaptoalkylsiloxanes). Oligonucleotides terminated with 5' or 3' thionucleosides can also be used to attach oligonucleotides to solid surfaces. The following references describe other methods that can be used to attach oligonucleotides to nanoparticles: Nuzzo et al., J.Am.Chem.Soc., 109, 2358 (1987) (disulfides on gold); Allara and Tompkins, Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins, J.Colloid Interface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Iler, The Chemistry Of Silica, Chapter 6, (Wiley 1979) (Carboxylic Acids on Silica); Timmons and Zisman, J.Phys.Chem., 69, 984-990 (1965) (Carboxylic Acids on Platinum); Soriaga and Hubbard, J .Am.Chem.Soc., 104,3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc.Chem.Res., 13, 177 (1980) (sulfolane, sulfoxide and other functionalized compounds on platinum solvents); Hickman et al., J.Am.Chem.Soc., 111, 7271 (1989) (isocyanide on platinum); Maoz and Sagiv, Langmuir, 3, 1045 (1987) (silane on silica); Maoz and Sagiv, Langmuir, 3, 1034 (1987) (silane on silica); Wasserman et al., Langmuir, 5, 1074 (1989) (silane on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987) (titanium dioxide and aromatic carboxylic acids, aldehydes, alcohols and methoxyl groups on silica); Lee et al., J. Phys. Chem., 92, 2597 (1988) (rigid phosphates on metals).
g.通用探针g. Universal probe
在一些实施方案中，条形码寡核苷酸和多孔颗粒是通用探针的成员，其可以用于对包括至少两个部分的任何目标核酸的测定中。该“通用探针”包括单个“捕获”序列的寡核苷酸，该序列与报告寡核苷酸(例如，条形码DNA)的至少一部分互补，以及与目标识别寡核苷酸的一部分互补。目标识别寡核苷酸包括具有至少两个部分的序列；第一个部分包括对连接到多孔颗粒的捕获序列的互补序列，第二个部分包括对特定目标核酸序列的第一个部分的互补序列。各种类型的目标识别寡核苷酸与通用探针一起使用能获得很大的优势，使得可以转换或互换目标识别寡核苷酸文库，以便选择特定测试溶液中的特定目标核酸序列。将包括与目标核酸的第二个部分互补的序列的捕获寡核苷酸连接到磁性探针颗粒。In some embodiments, barcoded oligonucleotides and porous particles are members of a universal probe that can be used in assays for any target nucleic acid comprising at least two moieties. The "universal probe" comprises an oligonucleotide of a single "capture" sequence that is complementary to at least a portion of a reporter oligonucleotide (eg, barcode DNA) and a portion of a target recognition oligonucleotide. The target recognition oligonucleotide comprises a sequence having at least two parts; a first part comprising the complementary sequence to the capture sequence attached to the porous particle and a second part comprising the complementary sequence to the first part of the specific target nucleic acid sequence . The use of various types of target-recognizing oligonucleotides with universal probes can be used to great advantage so that libraries of target-recognizing oligonucleotides can be switched or interchanged in order to select a specific target nucleic acid sequence in a specific test solution. A capture oligonucleotide comprising a sequence complementary to the second portion of the target nucleic acid is attached to the magnetic probe particle.
为了提高的优势可以操纵这些通用探针，其取决于待进行的特定测定。通过简单地取代或互换目标识别寡核苷酸，使得通用探针的第二个部分包括对不同目标核酸的互补序列，探针可以“调向”各种单个目标核酸序列。相似地，如果要在单个测试溶液中测定多个目标核酸序列，报告寡核苷酸可以包括对每个目标核酸特异性的序列，借此，已知的特定序列的报告寡核苷酸的检测将表示测试溶液中特定目标核酸的存在。将包括与目标核酸的第二个部分互补的序列的捕获寡核苷酸连接到纳米颗粒。These universal probes can be manipulated to increased advantage, depending on the particular assay to be performed. By simply substituting or interchanging the target-recognizing oligonucleotides so that the second portion of the universal probe includes the complementary sequence to a different target nucleic acid, the probe can be "tuned to" a variety of individual target nucleic acid sequences. Similarly, if multiple target nucleic acid sequences are to be assayed in a single test solution, the reporter oligonucleotides can include sequences specific for each target nucleic acid, whereby detection of reporter oligonucleotides of known specific sequence will indicate the presence of a specific target nucleic acid in the test solution. A capture oligonucleotide comprising a sequence complementary to the second portion of the target nucleic acid is attached to the nanoparticle.
h.树状分子h. Dendrimers
在本发明该实施方案的一个方面中，提供了与用至少两种类型的寡核苷酸标记的树状分子缀合的颗粒。树状分子是由多个分支单元单体构成的结构，并用于各种应用中。参见，例如，Barth等，Bioconjugate Chemistry 5：58-66(1994)；Gitsov & Frechet，Macromolecules 26：6536-6546(1993)；Lochmann等，J.Amer.Chem.Soc.115：7043-7044(1993)；Miller等，J.Amer.Chem.Soc.114：1018-1025(1992)；Mousy等，Macromlecules 25：2401-2406(1992)；Naylor等，J.Amer.Chem.Soc.111：2339-2341(1989)；Spindeler & Frechet，Macromolecules 26：4809-4813(1993)；Turner等，Macromolecules，26：4617-4623(1993)；Wiener等，MagneticResonance Med.31(1)：1-8(1994)；Service，267：458-459(1995)；Tomalia，Sci.Amer.62-66(1995)；和Tomalia的U.S.专利No.4,558,120；4,507,466；4,568,737；4,587,329；4,857,599；5,527,524；5,338,532，以及Nilsen的U.S.专利No.6,274,743，全部以其整体通过引用并入本文，用于所有目的。树状分子提供了优于其他类型的超分子结构的重要优势，如接触最大体积最少结构单元，更容易控制大小，重量和生长特性的能力，并且可以衍生化多个末端来产生高度标记的分子，其在标记之间具有限定的空间，或提供用于其他分子连接的位点，或其组合。概括地参见U.S.专利No.6,274,723和上述关于合成方法的参考文献。用于本发明方法中的核酸树状分子是本领域已知的可以用核酸官能化的或从核酸/寡核苷酸产生的那些中的任何一种。可以根据公开文献合成这样的树状分子，如Hudson等，“Nucleic Acid Dendrimers：Novel Biopolymer Structures”Am.Chem.Soc.115：2119-2124(1993)；U.S.专利No.6,274,723；和Cantor的U.S.专利No.5,561,043。In an aspect of this embodiment of the invention there is provided a particle conjugated to a dendrimer labeled with at least two types of oligonucleotides. Dendrimers are structures composed of multiple branch unit monomers and are used in a variety of applications. See, e.g., Barth et al., Bioconjugate Chemistry 5:58-66 (1994); Gitsov & Frechet, Macromolecules 26:6536-6546 (1993); Lochmann et al., J.Amer.Chem.Soc.115:7043-7044 (1993) ); Miller et al., J.Amer.Chem.Soc.114:1018-1025 (1992); Mousy et al., Macromlecules 25:2401-2406 (1992); Naylor et al., J.Amer.Chem.Soc.111:2339- 2341 (1989); Spindeler & Frechet, Macromolecules 26: 4809-4813 (1993); Turner et al., Macromolecules, 26: 4617-4623 (1993); Wiener et al., Magnetic Resonance Med. 31 (1): 1-8 (1994) ; Service, 267:458-459 (1995); Tomalia, Sci.Amer.62-66 (1995); and U.S. Patent Nos. 4,558,120 of Tomalia; 4,507,466; Patent No. 6,274,743, which is hereby incorporated by reference in its entirety for all purposes. Dendrimers offer important advantages over other types of supramolecular structures, such as access to the largest volume of the fewest building blocks, the ability to more easily control size, weight, and growth properties, and the ability to derivatize multiple ends to produce highly labeled molecules , which have defined spaces between labels, or provide sites for attachment of other molecules, or a combination thereof. See generally U.S. Patent No. 6,274,723 and the above references for synthetic methods. Nucleic acid dendrimers for use in the methods of the invention are any of those known in the art that can be functionalized with nucleic acids or generated from nucleic acids/oligonucleotides. Such dendrimers can be synthesized according to published literature, such as Hudson et al., "Nucleic Acid Dendrimers: Novel Biopolymer Structures" Am. Chem. Soc. 115:2119-2124 (1993); U.S. Patent No. 6,274,723; and Cantor's U.S. Patent No. 5,561,043.
IV.比色方法IV. Colorimetric method
在优选的实施方案中，本发明提供了简单而超灵敏的比色生物条形码测定。本发明的筛选方法和检测方案基于发明人之一和其他人在以下参考文献中所述的那些：US专利申请No.10/877,750，以US20050037397公开；U.S.专利申请No.10/788,414，以US20050009206公开；和U.S.专利申请No.10/108211，以US20020192687公开，在此再次将全部通过引用并入本文，用于所有目的。在优选的实施方案中，通过提供具有提高的生物条形码DNA扩增以及定量和多路能力的比色测定，本发明的生物条形码测定提供了改进的分析物检测的生物条形码方法。In a preferred embodiment, the present invention provides a simple and ultrasensitive colorimetric biological barcoding assay. The screening methods and detection protocols of the present invention are based on those described by one of the inventors and others in the following references: US Patent Application No. 10/877,750, published as US20050037397; U.S. Patent Application No. 10/788,414, published as US20050009206 publication; and U.S. Patent Application No. 10/108211, published as US20020192687, which is hereby incorporated by reference in its entirety for all purposes. In preferred embodiments, the biological barcoding assays of the present invention provide improved biological barcoding methods for analyte detection by providing a colorimetric assay with enhanced biological barcoding DNA amplification as well as quantification and multiplexing capabilities.
在一个实施方案中，如实施例中所示的，使用比色测定来检测条形码DNA，因为它不需要复杂的装置或实验步骤。探针溶液的简单混合和分离将导致渺摩尔灵敏度，而不需要使用微阵列、复杂的信号扩增步骤如酶扩增和银加强或精密复杂的信号测量工具。因为读出是基于颜色改变，进行测定只需要最少的专门技术。In one embodiment, as shown in the Examples, a colorimetric assay is used to detect barcoded DNA since it does not require complicated equipment or experimental steps. Simple mixing and separation of probe solutions will result in attomolar sensitivity without the use of microarrays, complex signal amplification steps such as enzyme amplification and silver enhancement, or sophisticated signal measurement tools. Because the readout is based on a color change, minimal expertise is required to perform the assay.
在一些实施方案中，可使用图像分析工具检测和定量颜色改变。在另一个实施方案中，通过眼睛目测颜色改变。In some embodiments, image analysis tools can be used to detect and quantify color changes. In another embodiment, the color change is visualized by eye.
在一些实施方案中，通过比色测定进行条形码寡核苷酸的检测。在一些实施方案中，比色测定包括通过以下方法来检测条形码寡核苷酸：提供包括第一和第二颗粒探针的溶液，其中第一颗粒探针包括与条形码寡核苷酸的一个末端互补的捕获寡核苷酸，并且其中第二颗粒探针包括与条形码寡核苷酸的另一相对末端互补的捕获寡核苷酸；将条形码寡核苷酸接触溶液并使条形码寡核苷酸与第一和第二颗粒探针杂交，借此第一和第二颗粒探针聚集成聚集物，其中溶液中的颜色改变表示所述聚集物的形成；并且检测所述溶液中的颜色改变。In some embodiments, detection of barcoded oligonucleotides is performed by a colorimetric assay. In some embodiments, the colorimetric assay comprises detecting a barcode oligonucleotide by providing a solution comprising a first and a second particle probe, wherein the first particle probe comprises an end of the barcode oligonucleotide a complementary capture oligonucleotide, and wherein the second particle probe comprises a capture oligonucleotide complementary to the other opposite end of the barcode oligonucleotide; contacting the barcode oligonucleotide with the solution and causing the barcode oligonucleotide to hybridizing to the first and second particle probes whereby the first and second particle probes aggregate into aggregates, wherein a color change in solution indicates formation of the aggregates; and detecting the color change in solution.
通过对聚集的纳米颗粒的视觉检测进行条形码DNA的比色检测。每种类型的纳米颗粒含有与对特定目标分析物的特异性条形码寡核苷酸互补的预定捕获寡核苷酸。在目标分析物的存在下，作为微粒，磁性颗粒和目标分析物之间的结合相互作用的结果，产生了探针复合物。从复合物中释放出条形码寡核苷酸，并可以通过任何合适的方式例如热变性来分离和分析，来检测一种或多种不同类型的报告寡核苷酸的存在。然而，考虑到对于比色检测，进一步的扩增不是必需的。Colorimetric detection of barcoded DNA was performed by visual detection of aggregated nanoparticles. Each type of nanoparticle contains a predetermined capture oligonucleotide complementary to a specific barcode oligonucleotide for a particular target analyte. In the presence of the target analyte, a probe complex is generated as a result of the binding interaction between the microparticle, the magnetic particle and the target analyte. The barcoded oligonucleotides are released from the complex and can be isolated and analyzed by any suitable means, such as heat denaturation, to detect the presence of one or more different types of reporter oligonucleotides. However, consider that for colorimetric detection, further amplification is not necessary.
在优选的实施方案中，该方法进一步包括有效使寡核苷酸之间发生特异性结合相互作用以形成聚集复合物的条件下，将含有颗粒捕获探针的溶液接触条形码寡核苷酸，以发出样品中存在目标分析物的信号；检测颜色改变的存在或不存在。在一个实施方案中，在步骤中使用颗粒探针来检测从探针复合物中分离出来的条形码DNA。In a preferred embodiment, the method further comprises contacting the solution containing the particle capture probe with the barcoded oligonucleotide under conditions effective to allow specific binding interactions between the oligonucleotides to form an aggregation complex, to Signals the presence of the analyte of interest in the sample; detects the presence or absence of a color change. In one embodiment, a particle probe is used in the step to detect barcoded DNA isolated from the probe complex.
本发明优选用于检测核酸的是金或银纳米颗粒。金和银胶体颗粒对于产生其漂亮颜色的条带具有高消光系数。这些强烈的颜色随着颗粒大小，浓度，颗粒间的距离以及聚集程度和聚集物形状(几何学)而改变，使这些材料对于比色测定特别有吸引力。例如，连接金纳米颗粒的寡核苷酸与寡核苷酸和核酸的杂交形成肉眼可见的立即颜色改变(参见，例如，实施例和图4B)。合适的纳米颗粒也可以从例如TedPella，Inc.(金)，Amersham Corporation(金)和Nanoprobes，Inc.(金)购得。Preferred for use in the present invention in the detection of nucleic acids are gold or silver nanoparticles. Gold and silver colloidal particles have high extinction coefficients for producing bands of their beautiful colors. These intense colors vary with particle size, concentration, distance between particles, and degree of aggregation and aggregate shape (geometry), making these materials particularly attractive for colorimetric assays. For example, hybridization of gold nanoparticle-attached oligonucleotides to oligonucleotides and nucleic acids results in an immediate color change visible to the naked eye (see, eg, Examples and Figure 4B). Suitable nanoparticles are also commercially available from, for example, TedPella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
ChadA.Mirkin，Robert L.Letsinger，Robert C.Mucic，JamesJ.Storhoff也已经描述过使用这样的纳米颗粒用于比色检测的方法，A DNA-based method for rationally assembling nanoparticles intomacroscopic materials，Nature 382，607-609(1996年8月15日)和Selective Colorimetric Detection of Polynucletides Based onthe Distance-Dependent Optical Properties of GoldNanoparticles，Science 22，1999年8月，277：1078-1081。在使用金纳米颗粒探针的优选实施方案中，观察到颜色从红色变为紫色。Chad A. Mirkin, Robert L. Letsinger, Robert C. Mucic, James J. Storhoff have also described methods using such nanoparticles for colorimetric detection, A DNA-based method for rationally assembling nanoparticles into macroscopic materials, Nature 382, 607 -609 (August 15, 1996) and Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles, Science 22, August 1999, 277:1078-1081. In a preferred embodiment using gold nanoparticle probes, a color change from red to purple was observed.
参照图4B，该方法可以是多路的。在此多路指的是同时检测一种溶液中的许多不同目标。可以按照图4A中所示的进行该多路技术。一种纳米结构(例如，13nm金纳米颗粒)可以和不同的点位置一起使用(这是较简单的形式)。然而，使用多个标记的多路技术将更有益(这是真正的多路，因为通过进行一个实验可以从一个试管检测几个标记，并且通过看颜色读出可以区分目标)。在此的主要构思是使用呈现不同光学特性的不同纳米结构(形状，组成和大小是变量)，这些特性使得可以用呈现出许多不同颜色的不同纳米结构来标记目标分子。Referring to Figure 4B, the method can be multiplexed. Multiplexing here refers to the simultaneous detection of many different targets in one solution. This multiplexing can be performed as shown in Figure 4A. One nanostructure (eg, 13nm gold nanoparticles) can be used with different spot positions (this is the simpler form). However, multiplexing with multiple markers would be more beneficial (this is true multiplexing because several markers can be detected from one tube by performing one experiment and targets can be distinguished by looking at the color readout). The main idea here is to use different nanostructures exhibiting different optical properties (shape, composition and size are variables) that make it possible to label target molecules with different nanostructures exhibiting many different colours.
再一次参照图4B，还可以使用银纳米颗粒和其他用于读出的量子点进行该方法。在使用银纳米颗粒探针的实施方案中，颜色改变可以从橙色，黄色或绿色并取决于颗粒的大小、形状等通常变为更深的黄色或绿色。Referring again to Figure 4B, the method can also be performed using silver nanoparticles and other quantum dots for readout. In embodiments using silver nanoparticle probes, the color change can be from orange, yellow or green to typically a darker yellow or green depending on the size, shape, etc. of the particle.
a.条形码寡核苷酸的比色检测a. Colorimetric detection of barcoded oligonucleotides
一旦DNA条形码或报告寡核苷酸通过去杂交从探针复合物中的多孔微粒中释放出来，可以通过任何合适的方法来检测。通常，在检测之前，DNA条形码通过去杂交从复合物中释放出来。可以使用任何合适的溶液或介质，将DNA条形码去杂交并从复合物中释放出来。代表性的介质是水。Once the DNA barcode or reporter oligonucleotide is released from the porous microparticle in the probe complex by dehybridization, it can be detected by any suitable method. Typically, DNA barcodes are released from the complex by dehybridization prior to detection. The DNA barcodes can be dehybridized and released from the complex using any suitable solution or medium. A representative medium is water.
在优选的实施方案中，通过以下的方法来检测条形码DNA：(a)提供包括第一和第二纳米颗粒探针的溶液，其中用与所述条形码寡核苷酸的所述特异性DNA序列的一个末端互补的捕获寡核苷酸官能化第一纳米颗粒探针，并且其中用与所述条形码寡核苷酸的所述特异性DNA序列的另一相对末端互补的捕获寡核苷酸官能化第二纳米颗粒探针；(b)将从探针复合物中分离出来的所述条形码寡核苷酸与所述溶液混合，以允许所述条形码寡核苷酸与所述纳米颗粒探针杂交和所述纳米颗粒探针聚集物的装配，其中溶液中的颜色改变反映出所述聚集物的形成；(c)将所述溶液点样在基质(substrate)上；(d)检测与对照相比较所述溶液中的颜色改变。In a preferred embodiment, barcoded DNA is detected by (a) providing a solution comprising first and second nanoparticle probes, wherein said specific DNA sequence associated with said barcoded oligonucleotide The first nanoparticle probe is functionalized with a capture oligonucleotide complementary to one end of the barcode oligonucleotide, and wherein the capture oligonucleotide is functionalized with the other opposite end of the specific DNA sequence of the barcode oligonucleotide. (b) mixing the barcode oligonucleotide separated from the probe complex with the solution to allow the barcode oligonucleotide to interact with the nanoparticle probe hybridization and assembly of said nanoparticle probe aggregates, wherein a color change in solution reflects the formation of said aggregates; (c) spotting said solution on a substrate; (d) detecting and comparing The color change in the solutions was compared photographically.
在另一个实施方案中，可以通过使用具有与其结合或连接的纳米颗粒探针的基质来扩大可检测的改变(信号)和提高测定的灵敏度。然后将含有条形码寡核苷酸的溶液置于基质上，用于随后的检测。In another embodiment, the detectable change (signal) can be amplified and the sensitivity of the assay improved by using a substrate with nanoparticle probes bound or linked thereto. The solution containing the barcoded oligonucleotides is then placed on the substrate for subsequent detection.
在优选的实施方案中，在溶液中提供了用与所述特异性DNA序列的一部分互补的捕获寡核苷酸官能化的纳米颗粒探针。提供了两组纳米颗粒探针；各自用与从探针复合物释放出来的条形码寡核苷酸的特异性DNA序列的两个末端之一互补的捕获寡核苷酸官能化。因此，连接到一组纳米颗粒探针的捕获寡核苷酸具有与待检测的条形码寡核苷酸的序列5’端互补的序列，而另一组纳米颗粒探针具有与待检测的条形码寡核苷酸的序列3’端互补的序列。然后在有效允许纳米颗粒探针上的捕获寡核苷酸与条形码寡核苷酸杂交的条件下，将条形码寡核苷酸接触两组纳米颗粒探针。以这种方式，条形码寡核苷酸与至少两个纳米颗粒探针结合，以允许纳米颗粒探针聚集物的装配。由此纳米颗粒探针聚集物的形成反映在含有捕获纳米颗粒探针聚集物的溶液的比色改变中。然后可以将溶液点样或传送至基质上，用于随后的检测。In a preferred embodiment, nanoparticle probes functionalized with capture oligonucleotides complementary to a portion of said specific DNA sequence are provided in solution. Two sets of nanoparticle probes are provided; each functionalized with a capture oligonucleotide complementary to one of the two ends of the specific DNA sequence of the barcode oligonucleotide released from the probe complex. Thus, the capture oligonucleotides attached to one set of nanoparticle probes have a sequence that is complementary to the 5' end of the sequence of the barcode oligonucleotide to be detected, while the other set of nanoparticle probes have a sequence that is complementary to the sequence of the barcode oligonucleotide to be detected. A sequence of nucleotides that is complementary to the 3' end. The barcode oligonucleotides are then exposed to the two sets of nanoparticle probes under conditions effective to permit hybridization of the capture oligonucleotides on the nanoparticle probes to the barcode oligonucleotides. In this way, barcoded oligonucleotides are bound to at least two nanoparticle probes to allow assembly of nanoparticle probe aggregates. The formation of nanoparticle probe aggregates is thus reflected in the colorimetric change of the solution containing the captured nanoparticle probe aggregates. The solution can then be spotted or delivered onto the substrate for subsequent detection.
如果溶液中存在足量的复合物，可以使用或不使用背景基质肉眼观察复合物。可以使用任何允许观察可检测改变的基质。合适的基质包括透明的固体表面(例如，玻璃，石英，塑料和其他聚合物)，不透明的固体表面(例如，白色的固体表面，如TLC硅石板，滤纸，玻璃纤维滤器，硝酸纤维素膜，尼龙膜)，和导电性固体表面(例如，铟-锡-氧化物(ITO))。基质可以是任何形状或厚度，但是通常是平而薄的。优选的是透明基质，如玻璃(例如，玻璃载玻片)或塑料(例如，微量滴定平板的孔)。在优选的实施方案中，基质是TLC板。Complexes can be visualized with or without background matrix if sufficient complexes are present in solution. Any substrate that allows observation of detectable changes can be used. Suitable substrates include transparent solid surfaces (e.g., glass, quartz, plastic, and other polymers), opaque solid surfaces (e.g., white solid surfaces such as TLC silica plates, filter paper, glass fiber filters, nitrocellulose membranes, nylon membrane), and conductive solid surfaces (eg, indium-tin-oxide (ITO)). The matrix can be of any shape or thickness, but is usually flat and thin. Preferred are transparent substrates, such as glass (eg, glass slides) or plastic (eg, wells of a microtiter plate). In a preferred embodiment, the substrate is a TLC plate.
在一个实施方案中，在比色改变的检测用于患者疾病状态诊断时，为确保对抗假阳性发生率，应当提供多个板组或阵列用于测试。例如，提供了高通量微平板，含有多个孔，每个孔含有相同的特定条形码和磁性颗粒探针的溶液来鉴定目标分析物。在另一个实施方案中，对于每个单个标记或分析物，将该方法的检测步骤进行多次。例如，临床医生将形成五个点用于条形码分析，除去最高和最低点强度的点，并使用其他三个点用于最终的定量和诊断。In one embodiment, to insure against the incidence of false positives when the detection of colorimetric changes is used for the diagnosis of a disease state in a patient, multiple plate sets or arrays should be provided for testing. For example, high-throughput microplates are provided, containing multiple wells each containing the same specific barcode and a solution of magnetic particle probes to identify target analytes. In another embodiment, the detection step of the method is performed multiple times for each single marker or analyte. For example, a clinician would form five spots for barcode analysis, remove the spots with the highest and lowest spot intensities, and use the other three spots for final quantitation and diagnosis.
还考虑为了检测提供的两组纳米颗粒探针可以是相同或不同类型的纳米颗粒。这可以进一步允许多路技术，用于鉴定样品中存在的一个或多个至许多个不同目标分析物的目的。参照图4A，使用多个标记的多路技术将更有利，允许检测一个样品孔中的几个目标分析物。使用不同纳米颗粒混合物的多路技术可能需要使用瑞利光散射或拉曼光谱学的检测，用于检测每个纳米颗粒的特异性光学特征或波长，如本领域中已知并实践的。It is also contemplated that the two sets of nanoparticle probes provided for detection may be the same or different types of nanoparticles. This may further allow multiplexing for the purpose of identifying one or more to many different target analytes present in a sample. Referring to Figure 4A, multiplexing using multiple labels would be more advantageous, allowing detection of several target analytes in one sample well. Multiplexing techniques using mixtures of different nanoparticles may require detection using Rayleigh light scattering or Raman spectroscopy for detecting specific optical features or wavelengths of each nanoparticle, as known and practiced in the art.
本发明还包括提供用于检测样品中存在的多于一个目标分析物的阵列。例如，提供含有多个孔的高通量微平板，每个孔具有含有鉴定目标分析物的特异性探针的溶液。The present invention also includes providing an array for detecting more than one target analyte present in a sample. For example, high-throughput microplates are provided that contain multiple wells, each well having a solution containing a specific probe that identifies an analyte of interest.
在另一个实施方案中，使用微射流技术来自动化和制备大规模平行的阵列。合适的微射流装置可以基于发明人之一和其他人在Proc.Natl.Acsd.Sci.USA，102，9745(2005)中所描述的，在此以其整体通过引用并入本文。In another embodiment, microfluidics is used to automate and fabricate massively parallel arrays. A suitable microfluidic device may be based on that described by one of the inventors and others in Proc. Natl. Acsd. Sci. USA, 102, 9745 (2005), which is hereby incorporated by reference in its entirety.
现在参照图2，本发明进一步提供了用于定量比色条形码DNA检测测定的定量方法，这使用之前的基于金纳米颗粒的比色DNA检测方案是不可能的。可以使用图形软件进行该定量方法，该软件是使用包括以下步骤的方法开发的：(a)获取基质上聚集物点的数字图像；(b)选择点用于分析；(c)计算与对照点相比较的点强度。在一个实施方案中，步骤(b)进一步包括调节对比度的步骤，用于更好的显现观察和表征。在优选的实施方案中，在使用金纳米颗粒的情况中，根据以下计算聚集物的定量，并因此计算出样品中存在的分析物的量：Referring now to Figure 2, the present invention further provides a quantitative method for quantitative colorimetric barcoded DNA detection assays, which was not possible using previous gold nanoparticle based colorimetric DNA detection schemes. This quantitative method can be performed using graphics software developed using a method comprising: (a) acquiring digital images of aggregate spots on the substrate; (b) selecting spots for analysis; (c) counting and comparing spots Compare point intensities. In one embodiment, step (b) further comprises the step of adjusting the contrast for better visual observation and characterization. In a preferred embodiment, where gold nanoparticles are used, the quantification of aggregates, and thus the amount of analyte present in the sample, is calculated according to:
点强度与条形码DNA寡核苷酸的数量成比例，即，越多的纳米颗粒聚集，越少的红色出现；并且条形码DNA寡核苷酸的数量与存在的目标蛋白的量成比例。The spot intensity is proportional to the number of barcoded DNA oligonucleotides, ie, the more nanoparticles are aggregated, the less red color appears; and the number of barcoded DNA oligonucleotides is proportional to the amount of target protein present.
在优选的实施方案中，将条形码DNA加入金纳米颗粒探针后，将溶液点样于TLC板上并干燥。扫描平板来获取平板的数字扫描。使用图形程序如ADOBE PHOTSHOP软件调节扫描图像对比度。然后选择每个纳米颗粒点，并使用定量函数如具有红色通道选项的PHOTOSHOP中的柱状图功能来定量选定的区域。使用来自柱状图窗口的平均值来计算每个点的点强度。In a preferred embodiment, after adding the barcoded DNA to the gold nanoparticle probes, the solution is spotted on a TLC plate and dried. Scan the flatbed to obtain a digital scan of the flatbed. Use a graphics program such as Adobe Photoshop software to adjust the contrast of the scanned image. Then select each nanoparticle spot and use a quantification function such as the histogram function in PHOTOSHOP with the red channel option to quantify the selected area. Use the mean value from the histogram window to calculate the spot intensity for each spot.
最后，该测定应当适用于护理点应用，只需要探针溶液和TLC板。目前正在进行优化检测系统用于更好的定量以及使系统多路化用于其他细胞因子的努力。考虑了可以根据探针溶液的浓度，探针大小，反应时间改变或优化所述的本发明的实施方案，合成更多单分散的多孔微粒，或通过最小化交叉反应性用于多路技术(例如，通过进一步的探针钝化或调节反应时间)。Finally, the assay should be suitable for point-of-care applications, requiring only a probe solution and a TLC plate. Efforts are currently underway to optimize the detection system for better quantitation and to multiplex the system for other cytokines. It is contemplated that the described embodiments of the invention can be varied or optimized depending on the concentration of the probe solution, probe size, reaction time, synthesis of more monodisperse porous microparticles, or use in multiplex techniques by minimizing cross-reactivity ( For example, by further probe deactivation or adjusting the reaction time).
V.检测分析物的试剂盒V. Kits for Detecting Analytes
在一个实施方案中，本发明提供了进行本发明方法的试剂盒，包括高通量微平板，含有孔阵列，每个孔含有相同或不同的特异性条形码和磁性颗粒探针的溶液，来鉴定目标分析物。将等份样品与阵列中的每个孔混合，因此允许在平行孔中进行测定。在另一个实施方案中，对于每个单个标记或分析物，将该方法的检测步骤进行多次。例如，临床医生将形成五个点用于条形码分析，除去最高和最低点强度的点，并使用其他三个点用于最终的定量和诊断。In one embodiment, the invention provides a kit for performing the method of the invention comprising a high-throughput microplate comprising an array of wells, each well containing the same or a different specific barcode and a solution of magnetic particle probes to identify target analyte. An aliquot of sample is mixed with each well in the array, thus allowing assays to be performed in parallel wells. In another embodiment, the detection step of the method is performed multiple times for each single marker or analyte. For example, a clinician would form five spots for barcode analysis, remove the spots with the highest and lowest spot intensities, and use the other three spots for final quantitation and diagnosis.
任选，在一个实施方案中，本发明提供了进行图像分析的装置，包括用于获得数字信号的工具，如平板扫描仪或CCD相机，和用于分析的工具，如具有可以分析像素强度的图形软件的计算机。在优选的实施方案中，该装置包括简单的平板扫描仪和具有如ADOBE PHOTOSHOP(Adobe Systems，San Jose，CA)软件的计算机来分析像素强度。Optionally, in one embodiment, the present invention provides means for performing image analysis, including means for obtaining digital signals, such as a flatbed scanner or a CCD camera, and means for analysis, such as having a sensor that can analyze pixel intensities Graphics software for computer. In a preferred embodiment, the device includes a simple flatbed scanner and a computer with software such as ADOBE PHOTOSHOP (Adobe Systems, San Jose, CA) to analyze pixel intensities.
VI.实施例VI. Embodiment
以下实施例用来举例和说明，但不限制本发明。The following examples serve to illustrate and illustrate, but not limit, the invention.
实施例1：材料和方法Example 1: Materials and methods
电子显微照相。使用了UC Berkeley Microlab机构的LEO 1550扫描电子显微镜(SEM)。在～3nm铬汽相沉积至样品上后，使用3kV加速电压以3mm的工作距离拍摄图像。Electron Micrography. A LEO 1550 scanning electron microscope (SEM) at the UC Berkeley Microlab facility was used. After ~3nm chromium vapor deposition onto the sample, images were taken using an accelerating voltage of 3kV at a working distance of 3mm.
条形码探针制备。为了制备条形码探针，将1ml氨基官能化的多孔硅石微粒(1.57×109ml-1直径：3.53±0.49μm；从Phenomenex，Torrance，CA获得)的水悬液在10,000rpm离心5分钟，并除去上清液。将颗粒重悬浮于PBS溶液中，并再一次重复离心步骤。将所得到的聚苯乙烯颗粒沉淀重悬浮于1ml pH7.4的PBS溶液中的8％戊二醛中。将溶液在摇摆振荡器上混合5小时。在10,000rpm离心5分钟，并丢弃上清液(将该步骤再重复两次)。将所得到的沉淀重悬浮于PBS中，并将5μg IL-2单克隆抗体加入溶液中。抗体的量(5μg)比Polysciences，Inc.推荐的用来完全修饰颗粒表面的抗体量低得多(抗体购自Abcam，Inc，Cambridge，MA)。将溶液置于振荡器上过夜，来将抗IL-2连接到活化的聚苯乙烯颗粒。其他人已经广泛使用类似的戊二醛连接物化学来实现蛋白质与氨基官能化颗粒的连接。然后将3’氨基官能化条形码DNA互补物(1ml 100μM；5’CGTCGCATTCAGGATTCTCAACTCGTAGCT-A10-C6-胺3’(SEQ ID NO：1))加至单克隆抗体修饰的硅石颗粒，并将离心步骤重复两次。将所得到的沉淀重悬浮于1ml 0.2M乙醇胺中，以在室温将微粒上所有未反应的戊二醛位点钝化30分钟。进行离心来除去上清液。随后加入牛血清白蛋白溶液(10％ BSA)来进一步钝化颗粒表面的蛋白无活性区域。将离心步骤重复两次，并除去上清液。将所得到的沉淀重悬浮于1ml0.15MPBS溶液中。Barcoded probe preparation. To prepare barcode probes, 1 ml of an aqueous suspension of amino-functionalized porous silica microparticles (1.57×109 ml−1 diameter: 3.53±0.49 μm; obtained from Phenomenex, Torrance, CA) was centrifuged at 10,000 rpm for 5 minutes, and Remove the supernatant. The pellet was resuspended in PBS solution and the centrifugation step was repeated once more. The resulting polystyrene pellet was resuspended in 1 ml of 8% glutaraldehyde in PBS solution, pH 7.4. The solution was mixed on a rocking shaker for 5 hours. Centrifuge at 10,000 rpm for 5 minutes and discard the supernatant (this step is repeated two more times). The resulting pellet was resuspended in PBS, and 5 µg of IL-2 monoclonal antibody was added to the solution. The amount of antibody (5 μg) was much lower than that recommended by Polysciences, Inc. to completely modify the particle surface (antibodies were purchased from Abcam, Inc, Cambridge, MA). The solution was placed on a shaker overnight to attach anti-IL-2 to the activated polystyrene particles. Similar glutaraldehyde linker chemistry has been widely used by others to link proteins to amino-functionalized particles. The 3' amino-functionalized barcode DNA complement (1
磁性探针制备。将氨基官能化的磁性颗粒(Dynal Biotech，BrownDeer，WI)连接到IL-2的单克隆抗体。这些抗体的表位不同于用于使用戊二醛-胺偶联化学制备条形码探针的抗体的那些(Abcam，Cambridge，MA)。用10ml吡啶洗涤缓冲液洗涤0.05mM EDTA溶液中的氨基官能化的磁性颗粒(5ml 1mg/ml的溶液)。将所得到的溶液磁性分离，并除去上清液(再重复两次)。然后在室温用5ml吡啶洗涤缓冲液中的5％戊二醛活化磁性颗粒3小时。然后将活化的磁性颗粒磁性分离，并除去上清液。将该磁性分离步骤重复两次，并将磁性颗粒重悬浮于10ml吡啶洗涤缓冲液中。然后将吡啶洗涤缓冲液中的单克隆抗IL-2(1ml750μg/ml)加入磁性颗粒中，并将溶液在室温混合10小时。然后，将1mg BSA加入磁性颗粒溶液中，并将溶液在室温再混合10小时。将磁性分离步骤重复两次，并将磁性颗粒重悬浮于5ml吡啶洗涤缓冲液中。然后将3ml甘氨酸溶液(1M，pH8.0)加入所得到的溶液中来淬灭所有未反应的醛位点，并将所得到的溶液搅拌30分钟。在磁性分离步骤后，将5ml洗涤缓冲液加入单克隆抗体官能化的磁性颗粒中并剧烈混合(将该步骤再重复两次)。然后将磁性颗粒磁性分离并除去上清液。将该洗涤步骤再重复三次。最后，将磁性探针重悬浮于0.15M PBS溶液中。Magnetic probe preparation. Amino-functionalized magnetic particles (Dynal Biotech, BrownDeer, WI) were linked to a monoclonal antibody to IL-2. The epitopes of these antibodies were different from those of the antibodies used to prepare barcode probes using glutaraldehyde-amine coupling chemistry (Abeam, Cambridge, MA). Amino-functionalized magnetic particles in a 0.05 mM EDTA solution (5 ml of a 1 mg/ml solution) were washed with 10 ml of pyridine wash buffer. The resulting solution was magnetically separated and the supernatant removed (repeated two more times). The magnetic particles were then activated with 5 ml of 5% glutaraldehyde in pyridine wash buffer for 3 hours at room temperature. The activated magnetic particles are then magnetically separated and the supernatant removed. This magnetic separation step was repeated twice and the magnetic particles were resuspended in 10 ml of pyridine wash buffer. Monoclonal anti-IL-2 (1 ml 750 μg/ml) in pyridine wash buffer was then added to the magnetic particles, and the solution was mixed at room temperature for 10 hours. Then, 1 mg BSA was added to the magnetic particle solution, and the solution was mixed for another 10 hours at room temperature. Repeat the magnetic separation step twice and resuspend the magnetic particles in 5 ml of pyridine wash buffer. Then 3 ml of glycine solution (1 M, pH 8.0) was added to the resulting solution to quench all unreacted aldehyde sites, and the resulting solution was stirred for 30 minutes. After the magnetic separation step, 5 ml of wash buffer was added to the mAb-functionalized magnetic particles and mixed vigorously (this step was repeated two more times). The magnetic particles are then magnetically separated and the supernatant removed. This washing step was repeated three more times. Finally, the magnetic probes were resuspended in 0.15M PBS solution.
条形码DNA定量。将条形码DNA加入金纳米颗粒探针后，将溶液点样在TLC板上并干燥。使用平板扫描仪扫描平板，并使用AdobePhotoshop软件调节扫描的图像(将所有的点一起调节)。然后选择每个纳米颗粒点，并使用Adobe Photoshop(Adobe SystemsIncorporated，San Jose，CA)具有红色通道选项的柱状图功能来定量选定的区域。使用来自柱状图窗口的平均值计算每个点的点强度(图2)。Barcoded DNA quantification. After adding the barcoded DNA to the gold nanoparticle probes, the solution was spotted on a TLC plate and dried. The flatbed was scanned using a flatbed scanner, and the scanned image was adjusted (all points together) using Adobe Photoshop software. Each nanoparticle spot was then selected and the selected area was quantified using the histogram function of Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) with the red channel option. Calculate the spot intensity for each spot using the mean value from the histogram window (Figure 2).
实施例2：细胞因子的比色生物条形码扩增测定Example 2: Colorimetric Biobarcoded Amplification Assay of Cytokines
在该工作中，我们的测定目标是白细胞介素-2(IL-2)。IL-2是分泌的人细胞因子蛋白质，其介导炎症和免疫应答过程中白细胞之间的局部相互作用。细胞因子在造血作用的调节中起着关键作用；介导表型上不同的细胞的分化，迁移，活化和增殖。21，22提高的细胞因子检测极限将使得可以更早和更准确地诊断和治疗癌症和免疫缺陷相关的疾病，并导致对细胞因子相关疾病和生物学的提高理解，因为当人受到外来抗原感染时，细胞因子是特征生物标记。常规的细胞因子检测测定具有～50fM的检测极限，而基于酶的滚动循环扩增方法的检测极限是～500aM。In this work, our assay target is interleukin-2 (IL-2). IL-2 is a secreted human cytokine protein that mediates local interactions between leukocytes during inflammation and immune responses. Cytokines play a key role in the regulation of hematopoiesis; mediating differentiation, migration, activation and proliferation of phenotypically distinct cells.21,22 Increased detection limits for cytokines will allow earlier and more accurate diagnosis and treatment of cancer and immunodeficiency-related diseases, and lead to an improved understanding of cytokine-related diseases and biology, because when a person is infected with a foreign antigen Cytokines are characteristic biomarkers. Conventional cytokine detection assays have a detection limit of -50 fM, whereas the detection limit of the enzyme-based rolling cycle amplification method is -500 aM.
在典型的生物条形码比色生物条形码测定中，制备了两种类型的探针(图1A)。第一种是条形码探针，用抗IL-2和寡核苷酸修饰的3μm多孔硅石颗粒，该寡核苷酸与条形码序列(5’AGCTACGAGTTGAGAATCCTGAATGCGACG3’(SEQ ID NO：2))互补，后者是目标分子的独特鉴定标签。第二种探针是2.8μm氧化铁磁性探针颗粒，其具有磁性氧化铁核心，具有胺修饰的硅烷涂层(Dynal Biotech，Brown Deer，WI)。用可以捕获IL2目标的抗IL-2分子官能化这些颗粒。In a typical biobarcoding colorimetric biobarcoding assay, two types of probes were prepared (Fig. 1A). The first is a barcode probe, a 3 μm porous silica particle modified with anti-IL-2 and an oligonucleotide complementary to the barcode sequence (5'AGCTACGAGTTGAGAATCCTGAATGCGACG3' (SEQ ID NO: 2)), the latter is a unique identification tag for the target molecule. The second probe is a 2.8 μm iron oxide magnetic probe particle with a magnetic iron oxide core with an amine-modified silane coating (Dynal Biotech, Brown Deer, WI). These particles are functionalized with anti-IL-2 molecules that can capture IL2 targets.
该测定的检测极限几个数量级优于其他的常规免疫测定。在一个实施方案中，测定在检测IL-2中表现出三个数量级更优(例如，在PBS缓冲液中30aM IL-2)。显著地，在该实施方案中，在检测IL-2中，该检测极限比基于酶的扩增方法灵敏～15倍。The detection limit of this assay is several orders of magnitude better than other conventional immunoassays. In one embodiment, the assay is three orders of magnitude better at detecting IL-2 (eg, 30 aM IL-2 in PBS buffer). Remarkably, in this embodiment, the detection limit is -15 times more sensitive than the enzyme-based amplification method in detecting IL-2.
在IL-2检测测定中(图1B)，将15μL磁性探针溶液(1.5×109珠子/ml)加入20μl IL-2溶液中，接着加入15μl条形码探针溶液(1×109珠子/ml)。将所得到的溶液在定轨振荡器上37℃孵育50分钟。接着，将溶液置于磁性分离器(Dynal Biotech，Brown Deer，WI)中，并除去上清液。然后，用0.15M PBS溶液再洗探针复合物溶液3次。最后，将50μl NANO纯水(18兆欧)加入磁性分离的复合物中来释放条形码DNA，并将复合物在70℃摇摆振荡器中保持10分钟。磁性分离后，收集包括游离条形码DNA链的上清液用于条形码DNA检测。为了检测条形码DNA，将30nm为条形码DNA捕获而官能化的金纳米颗粒探针(对于探针1和2均为25μl1nM)(条形码捕获探针1：5’TCTCAACTCGTAGCTAAAAAAAAAA-三甘醇-SH3’(SEQ ID NO：3)；条形码捕获探针2：5’SH-三甘醇-AAAAAAAAAACGTCGCATTCAGGAT3’(SEQ IDNO：4))加入0.15M PBS溶液中的条形码DNA。将所得到的溶液保持在室温一个半小时。然后将溶液离心来提高探针复合物的浓度和收集小的纳米颗粒聚集物(10,000rpm 5分钟)，并丢弃上清液。尽管在此使用了离心步骤，该步骤对于进一步优化后该测定的实际实施可能不是必需的。最后，将5μl来自浓缩的纳米颗粒溶液的纳米颗粒探针溶液点样在反相硅石TLC板(EMD Chemicals，Inc.，Gibbstown，NJ)上，用于目标证实和定量(图2A)。点测试为30aM至300fM，并包括其中不存在IL-2的对照样品。在背景蛋白(每个样品1μl5μM抗生物素和1μl5μM抗纤连蛋白)存在下，该测定可以检测低至30aMIL-2目标。点样的点不仅显示出不同的颜色，而且显示出不同的强度。使用基于反映出金纳米颗粒聚集的红色强度的图像分析软件(AdobePhotoshop，Adobe Systems Incorporated，San Jose，CA)定量每个点强度。因为该比色测定是基于颜色从红色(无条形码DNA)变为紫色(具有条形码DNA)，较低的平均红色通道值表示溶液中存在较多的条形码DNA(图2)。在此的点强度定义为对照点的平均红色通道值除以给定样品点的平均红色通道值。图3A中绘出了这些点强度值(将实验重复五次，并且最高和最低值没有用于最终的点强度计算)。30aM目标溶液的点强度高于对照点的点强度，并且该测定的动态范围为30aM至300fM(图3A)。In the IL-2 detection assay (Figure 1B), 15 μL of magnetic probe solution (1.5×109 beads/ml) was added to 20 μl of IL-2 solution, followed by 15 μl of barcode probe solution (1×109 beads/ml ). The resulting solution was incubated on an orbital shaker at 37°C for 50 minutes. Next, the solution was placed in a magnetic separator (Dynal Biotech, Brown Deer, WI), and the supernatant was removed. Then, the probe complex solution was washed again 3 times with 0.15M PBS solution. Finally, 50 μl of NANO pure water (18 megohm) was added to the magnetically separated complex to release the barcoded DNA, and the complex was kept on a 70° C. rocking shaker for 10 minutes. After magnetic separation, the supernatant including free barcoded DNA strands was collected for barcoded DNA detection. To detect barcoded DNA, a 30 nm gold nanoparticle probe functionalized for barcoded DNA capture (25
为了验证该比色生物条形码系统用于真实样品，使用用于PBS缓冲液中IL-2检测的相同方案测试了人血清样品中的IL-2分子(Cambrex Corp.，East Rutherford，NJ)。用于300aM，3fM，30fM和300fM IL-2样品的基于纳米颗粒的条形码检测点显著不同于对照点(图3B)。30fM后点强度快速饱和。To validate the use of this colorimetric biobarcoding system for real samples, IL-2 molecules were tested in human serum samples (Cambrex Corp., East Rutherford, NJ) using the same protocol used for IL-2 detection in PBS buffer. The nanoparticle-based barcode detection spots for 300aM, 3fM, 30fM and 300fM IL-2 samples were significantly different from control spots (Fig. 3B). After 30fM, the point intensity is rapidly saturated.
本说明中引用的任何专利，专利公开，出版物或GenBank登录号表示本发明所属领域技术人员的水平，并在此通过引用并入本文，至如同将每一篇特意和单独通过引用并入本文的相同程度。Any patents, patent publications, publications or GenBank accession numbers cited in this specification represent the level of skill in the art to which the invention pertains and are hereby incorporated by reference as if each were specifically and individually incorporated by reference to the same extent.
序列表sequence listing
<110>The Regents of the University of California<110>The Regents of the University of California
Nam，Jwa-MinNam, Jwa-Min
Groves，Jay T.Groves, Jay T.
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<223>连到该序列3′端的是“-三甘醇-SH”官能团<223> Attached to the 3' end of the sequence is the "-triethylene glycol-SH" functional group
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| CA2622719A1 (en) | 2007-07-26 |
| AU2006336290A2 (en) | 2008-07-10 |
| EP1931943A2 (en) | 2008-06-18 |
| WO2007084192A2 (en) | 2007-07-26 |
| AU2006336290A1 (en) | 2007-07-26 |
| US20080268450A1 (en) | 2008-10-30 |
| JP2009513948A (en) | 2009-04-02 |
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| WO2007084192A3 (en) | 2009-04-30 |
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